Method for expansion of tumour-reactive T-lymphocytes for immunotherapy of patients with specific cancer types

ABSTRACT

Methods for treating a patient suffering from a neoplastic disease are disclosed and described. A number of diseases can be treated under the present methods, including without limitation gall bladder cancer, hepato cellular cancer, ovarian cancer, small intestine cancer, lung cancer, mesothelioma, breast cancer, kidney cancer, pancreas cancer, prostate cancer, carcinoid cancer, leiomyosarcoma, or metastasis thereof. A general method for providing such treatment may include: 1) identifying in a patient one or more sentinel and/or metinel lymph nodes draining the neoplasm; 2) resecting the one or more nodes and, optionally all or part of the tumour or metastasis; 3) isolating tumour-reactive T-lymphocytes from said lymph nodes; 4) in vitro expanding said tumour-reactive T-lymphocytes; and 5) administering the thus obtained tumour-reactive T-lymphocytes to the patient.

PRIORITY CLAIM

This application claims the benefit of earlier filed U.S. ProvisionalApplication No. 60/937,484, filed Jun. 27, 2007, which is incorporatedherein by reference.

FIELD OF THE INVENTION

The invention relates to an improved method for expansion and activationof tumour-reactive lymphocytes, in particular CD4+ helper and/or CD8+T-lymphocytes. The T-lymphocytes are not CD4+ CD25+^(Hi) lymphocytes,i.e. the present invention does not cover regulatory T-lymphocytes. Thelymphocytes may be used for treating and/or preventing specific cancertypes, in particular gall bladder cancer, hepato cellular cancer,ovarian cancer, small intestine cancer, lung cancer, mesothelioma,breast cancer, kidney cancer, pancreas cancer, prostate cancer,carcinoid cancer, leiomyosarcoma, or metastasis thereof.

BACKGROUND OF THE INVENTION

According to the immune surveillance hypothesis, the immune system iscontinuously sensitized against developing tumours, where experimentalevidence strongly supports this notion. The identification of specifictumour antigens has created new possibilities for tumour immunotherapyand many immunotherapeutic approaches are now being translated intoclinical trials. Among these, adoptive transfer of tumourantigen-specific lymphocytes seems particularly promising. Theseattempts have, so far, usually been based on either mononuclear cellsfrom peripheral blood or tumour infiltrating lymphocytes (TIL) separatedfrom fresh tumour specimens. In recent trials, treatment of patientswith malignant melanoma with autologous transfer of expanded TILs,objective response rates of up to 51% has been reported. TIL cells arefew, they are frequently unresponsive (anergic) due to immunosuppressivemechanisms from the tumour creating long periods for expansions to occur(several months). Furthermore, the protocols have been aiming towardsthe expansion of CD8⁺ cytotoxic T cells and the cells have beenreintroduced into patients preconditioned with chemotherapy and inaddition the patients have been treated with high doses of interleukin-2to provide survival of CD8⁺ T cells.

Gallbladder Cancer

Gallbladder cancer is a relatively uncommon disease with approximately 9000 new cases per year in the United States. Gallbladder cancer is veryaggressive and commonly spreads to the liver, pancreas or stomach.

Patients diagnosed with gallbladder cancer face a grim prognosis withoverall survival being only 5%-10%. In cases where the cancer has spreadto other organs, long term survival is very rare.

The most common form of treatment today is surgical removal of thegallbladder and lymph node dissection.

Hepatocellular Carcinoma

Hepatocellular carcinoma is a malignant tumor of the liver. The diseaseaccounts for approximately 19 000 new cases each year in the UnitedStates. Infection with the Hepatitis B och C viruses has beenestablished as a major cause of the disease.

Prognosis of the disease is generally very poor. The most common form oftreatment is surgical resection of the tumor, in cases where possible.If the cancer cannot be removed, the disease is usually deadly within 3to 6 months.

Ovarian Cancer

Cancer of the ovaries accounts for approximately 27 000 new cases and 15000 deaths per year in the United States. There are over 25 major typesof ovarian neoplasms, the most common being serous adenocarcinoma.

Prognosis is very dependant upon in which stage the disease isdiagnosed. For early stages, long-term survival can reach over 90%.Survival is dramatically decreased with later diagnosis, and overallfive year survival for all stages is less than 40%.

Surgery is today the preferred form of treatment, with chemotherapy usedafter surgery to treat any residual disease.

Small Intestine Cancer

Small intestine cancer, or cancer of the small bowel, accounts for onlya minute proportion of all gastrointestinal malignancies. There areapproximately 6 000 new cases per year in the United States.Adenocarcinoma is the most common subtype of small intestine cancer,accounting for approximately half of the cases.

Inflammatory disease of the small bowel has been established as a majorrisk factor for small intestine cancer.

Surgery is today the only known effective treatment, and is possible inapproximately two thirds of all patients. Five year survival rates forpatients who undergo surgical resection of the tumor is 40-60%.

Breast Cancer

With almost 180 000 new cases per year, breast cancer is the most commontype of cancer among women in the United States, and the second mostcommon cause of cancer death (approximately 40 000 deaths per year).Over the last 50 years, the incidence of the disease has slowlyincreased.

Despite much research, the cause of breast cancer is still poorlyunderstood.

For staging and prognosis, evaluation of tumor cell presence in regionallymph nodes is commonly done in conjunction with surgery.

Along with surgery, treatment alternatives include chemotherapy,hormonal therapy, radiotherapy and immune therapy.

Lung Cancer

Carcinoma of the lung is the most common cause of cancer death worldwideand in the United States. More than 200 000 new cases are developed eachyear in the United States, and about 160 000 will die from the disease.

Cigarette smoking is by far the leading cause of lung cancer, accountingfor approximately 85% of all cases. All subtypes of lung cancer areassociated with cigarette smoking, but the strongest connection is withsquamos cell carcinoma and small cell carcinoma. In cases where thepatient is a non-smoker, the most common type is adenocarcinoma.

Patients with surgically resectable tumors have the best prognosis.Factors correlated with adverse prognosis include large tumor size andmetastases to lymph nodes.

Mesothelioma

Mesothelioma is a neoplasm of mesothelial cells, which is most common inthe pleura. Approximately 2 000 patients are affected each year in theUnited States.

More than 80% of patients are reported to have been exposed to asbestos.The latency period between exposure and appearance of the disease isusually 20-40 years.

There are no known effective treatments today, and the prognosis isdismal.

Kidney Cancer

The most common type of kidney cancer is renal cell carcinoma, followedby renal pelvis carcinoma. Each year, there are in total more than 50000 new cases of kidney cancer in the United States, and kidney canceris the cause of more than 12 000 deaths.

In patients where the cancer has not spread to other organs, kidneycancer can normally be cured with surgery. If the cancer has spread tolymph nodes or other organs, commonly the lung, bone or liver, long-termis reduced dramatically to only around 10%.

Prostate Cancer

Prostate cancer is a very common malignancy among older men. Patientsyounger than 50 years constitute less than 1% of all new cases (about220 000 each year) in the United States. Prostate cancer accounts forabout 25 000 deaths every year in the U.S.

Almost all of primary prostatic tumors are adenocarcinomas. The mostcommon metastatic sites are the lymph nodes, bonea, lung and liver.

Radiation therapy and surgical removal of the prostate are the mostcommon forms of treatment today.

Carcinoid Cancers

Carcinoid is an often malignant type of tumor originating in the cellsof the neuroendocrine system. The majority of carcinoid tumors are foundin the appendix, small intestine or rectum. The tumor can also originatein other locations, including the lungs, pancreas, bronchus, gallbladderovary and testis.

Surgical removal of the tumor is the standard treatment when possibleand is often curative. Carcinoid tumors are generally slow-growing. Inpatients with distant metastases, the median survival is about twoyears.

Leiomyosarcoma

Leiomyosarcoma is a rare type of malignant neoplasm of smooth muscle. Itcan arise almost anywhere in the body, but most commonly in the uterus,abdomen or pelvis.

Radiation therapy and chemotherapy are usually ineffective againstleiomyosarcoma. Surgical resection of the tumor can be curative. Thefive year survival rate for leiomyosarcoma is about 30%.

The immune system often appears informed about tumours, as shown by anaccumulation of immune cells at tumour sites, which correlates withimproved prognosis. Immuno-competent cells respond to “danger” signals,which can be provided of growing tumours as a consequence of thegenotoxic stress of cell transformation and disruption of thesurrounding microenvironment. Under ideal conditions, these signals willinduce inflammation, activate innate effector cells with antitumouractivity, and stimulate professional antigen-presenting cells (APCs),particularly dendritic cells (DCs), to engulf tumour-derived antigensand migrate to draining lymph nodes to trigger an adaptive response by Tand B lymphocytes. Thus, the immune system is capable of recognizing andeliminating tumour cells but unfortunately tumours often interfere withthe development and function of immune responses. However, recentadvances in cellular and molecular immunology suggest strategies, whichmay prevent antitumour responses. Briefly, the presence of a tumourindicates that the developing cancer was able to avoid detection or toescape or to overwhelm the immune response. Progressing tumours oftenexhibit strategies that promote evasion from immune recognition.Examples are physical exclusion of immune cells from tumour sites, poorimmunogenicity due to reduced expression of major histocompatibilitycomplex (MHC) or costimulatory proteins, and disruption of naturalkiller (NK) and natural killer T (NKT) cell recognition. Further, sometumours prevent triggering of an inflammatory response by secretingproteins such as interleukin 10 (IL-10) or vascular endothelial growthfactor (VEGF) that interfere with DC activation and differentiation, orby blocking the production of proinflammatory molecules by increasingexpression of the STAT3 protein. Even if a response is induced, tumourcells may escape elimination by losing targeted antigens, renderingtumour-reactive T cells anergic, inducing regulatory T cells, orspecifically deleting responding T cells. The tumour that finallydevelops reflects selection of poorly immunogenic and/orimmune-resistant malignant cells.

In the adjuvant setting, tumour immunotherapy offers an appealingalternative to traditional cytostatics. One strategy has been to expandand activate NK cells in vitro with out specific antigen by culture withIL-2 followed by infusion of large numbers of these NK cells back intopatients alone or with high doses of IL-2. This approach, oradministration of high doses of IL-2 to expand and activate NK cellsentirely in vivo, has yielded antitumour activity and remission in asubset of patients (Rosenberg S A et al., J Natl Cancer Inst 85, 622,1993). However, life-threatening toxicity often develops, largely due tothe release of tumour necrosis factor (TNF) from activated NK cells.

Thus, it is obvious that there is still a need for an effective and atthe same time safe treatment of gall bladder cancer, hepato cellularcancer, ovarian cancer, small intestine cancer, lung cancer,mesothelioma, breast cancer, kidney cancer, pancreas cancer, prostatecancer, carcinoid cancer, leiomyosarcoma, or metastasis thereof.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have previously shown that activation of naïve Tcells may occur within the highly specialized microenvironment ofsecondary lymphoid organs, such as the sentinel lymph node. In otherwords, the sentinel node may be regarded as the primary site for theimmune system to encounter tumour antigens.

The inventors have previously disclosed a general method for expansionof tumour-reactive T-lymphocytes from sentinel lymph nodes, showing thatit is possible to culture T-lymphocytes obtained from sentinel lymphnodes in order to obtain a culture of tumour-reactive T-lymphocytes. Thetumour-reactive T-lymphocytes may be used for treating cancer byadministering an effective amount of tumour-reactive T-lymphocytes tothe patient from which the sentinel nodes were removed.

The success of a cancer treatment comprising administration oftumour-reactive T-lymphocytes are determined by factors such as, e.g.,the amount of tumour-reactive T-lymphocytes obtained after the expansionstep, i.e. the amount of tumour-reactive T-lymphocytes available forinfusion to the patient, the time required to obtain an effective amountof tumour-reactive T-lymphocytes and the concentration and ratio ofspecific subpopulations of tumour-reactive T-lymphocytes obtained by theexpansion method.

Accordingly, the present invention discloses an improved method forexpansion of tumour-reactive CD4+ helper and/or CD8+ T-lymphocytes,wherein specific culturing conditions have been determined andoptimized, and wherein specific markers on the T-lymphocytes and in theculture medium are monitored throughout the expansion phase, in order toobtain high numbers of tumour-reactive T-lymphocytes in the shortestpossible time span. Furthermore, the invention at the same time providesa method for directing the development of tumour-reactive CD4+ helperand/or CD8+ T-lymphocytes towards specific subpopulations. TheT-lymphocytes are not CD4+ CD25+^(Hi) lymphocytes, i.e. the presentinvention does not cover regulatory T-lymphocytes.

CD4⁺ CD25^(Hi) T lymphocytes expressing the transcription factor FoxP3are considered regulatory T cells (Treg). Tregs have the property toregulate T helper and T cytotoxic cells by inhibiting activation andproliferation and in addition Treg inhibit the production and release ofuseful Th1 cytokines such as IFN-gamma. Thus, the method presented hereis developed in order to promote the expansion of Thelper cells and Tcytotoxic T cells and to avoid expansion of Treg cells.

The tumour-reactive T-lymphocytes most often generated by the presentmethod are CD4+ helper T-lymphocytes. One of the objects of the presentexpansion method is in some respect to imitate the natural pathway ofthe patient's own immune system, and to a certain degree let thecomponents of the patients immune system determine whether, in the firstplace, CD4+ helper or CD8+ T-lymphocytes are generated, depending onwhether antigen is presented by MCHI or MCHII. In most cases, theantigens will be presented by the class II MCH molecule leading togeneration of CD4+ helper T-lymphocytes. However, in some cases CD8+T-lymphocytes are generated. If CD4+ helper T-lymphocytes are generated,they will be further expanded as described herein, however, the methodmay also be used for expanding CD8+ cells.

The inventors have found that an expansion method comprising twodifferent phases are especially useful for obtaining a high number oftumour-reactive CD4+ helper and/or CD8+ T-lymphocytes in a relativelyshort time span, the two phases being

i) a first phase of stimulating tumour-reactive T-lymphocytes withtumour-derived antigen together with at least one substance havingagonistic activity towards the IL-2 receptor, to promote survival oftumour-reactive T-lymphocytes, and

ii) a second phase of activating and promoting growth of tumour-reactiveT-lymphocytes, wherein the second phase ii) is initiated when the CD25cell surface marker (IL-2R marker) is down-regulated on T-lymphocytes.

This expansion method may also be carried out using monocytes isolatedfrom the patient as antigen specific cells. The monocytes will beadministered to the patient when differentiated into dendritic cells bythe use of maturating cytokines such as IL-4, GM_CSF and IL-3 followedby activation of the dendritic cells by the addition of Toll likereceptor stimulating agencies such as lipopolysaccharide. The use ofmature activated dendritic cells as the antigen specific population maypromote and enhance the expansion of T helper cells and T cytotoxic Tcells.

DEFINITIONS

By the term “tumour-reactive T-lymphocytes” is intended to meanT-lymphocytes carrying a T cell receptor specific for and recognizing atumour antigen.

By the term “T helper cells” is intended to mean T-lymphocytes thatpromote adaptive immune responses when activated.

By the term “Th1 cells” is intended to mean T helper cells that promotecell mediated immune responses when activated, using cytokines such asIFN-gamma.

By the term “Th2 cells” is intended to mean T helper cells promotinghumoral immune responses when activated, using cytokines such as IL-4.

By the term “CD4+ helper T-lymphocytes” is intended to meanT-lymphocytes that express CD4 but not the transcription factor FoxP3.

By the term “CD8+ T-lymphocytes” is intended to mean T-lymphocytes thatexpress CD8.

By the term “regulatory T-lymphocyte” is intended to mean T-lymphocytesthat suppress adaptive immune responses, expressing transcription factorFoxP3.

By the term “specific activation” of T-lymphocytes is intended to meanantigen specific and MHC restricted T-cell receptor mediated activation.In contrast the term “unspecific activation” of T-lymphocytes isintended to mean a general activation of all T-cells, regardless ofT-cell receptor specificity.

The term “tumour-derived antigen” intends to cover tumour cells, ahomogenate of a tumour, which homogenate may be denatured, or tumourproteins, polypeptides or peptides, e.g. in the form of purified,natural, synthetic and/or recombinant protein, polypeptide or peptide.The tumour-derived antigen may be intact molecules, fragments thereof ormultimers or aggregates of intact molecules and/or fragments. Examplesof suitable polypeptides and peptides are such that comprises from about5 to about 30 amino acids, such as, e.g. from about 10 to 25 aminoacids, from about 10 to 20 amino acids or from about 12 to 18 aminoacids. If peptides are used, a final molar concentration in the cultureof from about 0.1 to about 5.0 μM, such as, e.g., from about 0.1 toabout 4.0 μM, from about 0.2 to about 3.0 μM, from about 0.3 to about2.0 μM or from about 0.3 to about 1.0 μM may be used. The tumour-derivedantigen may be autologous or heterologous, i.e. arise from the patientto be treated or be obtained from another subject suffering from cancer.In the present Examples the inventors uses an autologous denaturedtumour extract, however, as mentioned above, other sources of thetumour-derived antigen may also be feasible for use in a methodaccording to the invention.

By the term “day 1 of the first phase” or e.g. “day 5 of the secondphase” is to be understood the following: The day on which thelymphocytes are harvested is denoted day 0 (zero). Day 1 of the firstphase is defined as the day where the expansion is initiated by additionof at least one substance having agonistic activity towards the IL-2receptor, and maybe culture medium and/or tumour-derived antigen. Theexpansion phase i) may be initiated on day 0 (zero) or up till 2 daysafter harvest of the lymphocytes. The day on which the second phase isinitiated by addition of tumour-derived antigen is throughout the textdescribed as “day 1 of the second phase”.

By the term “sentinel lymph node” is intended to mean the first lymphnode(s) to receive lymphatic drainage from a tumour. The term “metinellymph node” refers to the first lymph node(s) to receive lymphaticdrainage from a metastasis.

By the term “V beta profile” or “V beta repertoire” is intended to meanthe distribution of groups of T cells expressing identical Vbeta domainsof the T cell receptor complex in a population of cells.

The first step in the present method is the identification of one ormore sentinel or metinel lymph nodes draining a tumour in a gall bladdercancer, hepato cellular cancer, ovarian cancer, small intestine cancer,lung cancer, mesothelioma, breast cancer, kidney cancer, pancreascancer, prostate cancer, carcinoid cancer, leiomyosarcoma, or metastasisthereof. The step i) of identifying one or more sentinel or metinelnodes are crucial for the method according to the invention, as thepresent inventors have shown that these nodes comprises a high amount ofT-lymphocytes activated against tumour-derived antigens in vivo.

One way of identifying the sentinel or metinel lymph node is byinjecting one or more lymph node locators into the patient, i.e. anysubstances suitable for locating a lymph node. Such locators arepreferably pharmaceutically acceptable and/or biocompatible. Thelocators can either be affinity based or non-affinity based. Examples ofaffinity based lymph node locators are antibodies in whole or fragments,nanobodies, nucleic acids such as RNA, DNA, and PNA all of which can bein turn labeled using various detection modalities. Detection ofaffinity based lymph node locators can be done by labeling with tracersand dyes, such as, e.g., the ones mentioned below. Visualization is thenmade by i) radiological methods such as x-ray, computerized tomography,scintigraphy, positron emission technique after labeling with contrastgenerating substances, such as, e.g., iodine containing substances orradioactive substances such as, e.g. technetium-99m, ii) magneticresonance imaging after labeling with magnetic or paramagneticsubstances, such as e.g., gadolinium, magnetodendromers or iron oxidecontaining particle; iii) light in the IR-visible-UV spectra by labelingwith dyes, fluorescent dyes or luminescent dyes for detection by thenaked eye or photon detecting devices such as CCD or CMOS sensors.

Examples of non-affinity based lymph node locators encompass tracers anddyes. These substances are transported in the lymph capillaries andaccumulate through phagocytosis by macrophages in the sentinel ormetinel node(s), thus identifying the tumour or metastasis draininglymph node(s).

Examples of tracers are radioactive substances such as, e.g.,technetium-99 for radioactive decay based detection with photonsensitive films or sensors such as PET detectors. Further on magnetic,paramagnetic or superparamagnetic substances, such as, e.g., gadoliniumcontaining contrast agents, iron oxide particles, magnetic oxideparticles, magnetodendrimers for magnetic resonance based detection,contrast agents, such as, e.g., iodine for radiological based detectionsuch as, e.g., computerized tomography or regular X-ray may be used.

Examples of dyes encompasses e.g., azo dyes, bisazo dyes, triazo dyes,diaryl methan dye, triaryl methan dye, anthrachino dye, polycyclicaromatic carbonyl dyes, indigo dyes for visualization by luminescence,near infrared, fluorescence, UV and visible light. Further on dyes alsoencompass luminescent substances for luminescence based detection andfluorescent substances, such as, e.g., pico green, sybr green, red Ooil, texas red for fluorescence based detection. Detection can dependingon the chosen wavelengths be made either by the naked eyes or photondetecting devices such as CCD or CMOS sensors.

In one embodiment the dye has an emission maximum that permitsvisualization by the naked eye in normal light. In another embodimentthe dye has an emission maximum that permits visualization by the nakedeye in UV light.

Other examples of suitable dyes or tracers appear from WO 04/045650,which is hereby incorporated by reference.

Another, but far more time-consuming way to identify sentinel or metinelnodes is to remove and investigate a selection of lymph nodes in thepresumed tumour or metastasis area. A tumour extract from the tumour ormetastasis of the actual patient could then be used to identify lymphnodes containing tumour-reactive T-lymphocytes by proliferating assays.

The lymph node locators are injected into the patient into, above,around, adjacent and/or under the tumour or metastasis. The locator willthen spread through lymph vessels leading into the metinel lymphnode(s), and the one or more nodes will start to get stained within acertain period of time, such as, e.g. within 5 min to 30 min, such as,e.g. within 5 min to 15 min after injection of the locator substance,where after the locator substance is imaged. As described above, imagingof the locator is of course dependent on the locator substance used.

If a dye having an emission maximum that allows visualization by thenaked eye in normal light is used, such as, e.g. Patent Blue, the one ormore sentinel or metinel nodes are simply identified as the nodes, whichare first to accumulate the coloured dye, i.e. if Patent Blue is used,the surgeon will look for the lymph nodes first to accumulate a bluecolour.

The locators may be injected by a single injection or by multipleinjections, such as, e.g., by two or more injections, by three or moreinjections, by four or more injections, by five or more injections or bysix or more injections.

How to perform the injections of the lymph node locators is dependent onthe location of the tumour or metastasis.

The lymph node locators may be injected involving a surgical procedure,i.e. a procedure that includes an incision. In such cases, the surgeonwill perform an incision in the area of the tumour and subsequently, alymph node locator may be injected directly into, above, around,adjacent and/or under the metastasis in order to identify the one ormore sentinel lymph nodes.

The lymph node locators may also be injected by a non-surgicalprocedure, i.e. a procedure that does not involve a surgical step,wherein a surgical step is defined a one including surgical operativeprocedures, i.e. involving incisions with an instrument. In the presentcontext, an injection, i.e. the punctuation of the skin with a needle,is not considered a surgical step. Accordingly, by the statement thatthe lymph node locator may be injected by a non-surgical procedure isintended to mean that the lymph node locator may be injected into,above, around, adjacent and/or under the metastasis directly into orthrough the skin.

Examples of situations wherein the lymph node locators may be injectedinto or through the skin, is e.g. cases where metastasis of tumours arelocated in the skin of the patient. In such situations the lymph nodelocator should preferentially be injected into the skin above themetastasis, or through the skin into, around, adjacent and/or under themetastasis. If the metastasis is located in the breast area of thepatient, the lymph node locator should preferentially be injected intothe skin above the metastasis, or through the skin into, around,adjacent and/or under the metastasis.

The metinel lymph nodes may not always be placed at the shortest or mostlogical anatomical distance from the metastasis. Accordingly, as ametinel lymph node may be placed distant from the metastasis, it may insome cases be very beneficial to inject a lymph node locator without theneed for surgery, as it may be very difficult to predict the position ofsuch a metinel lymph node, and there by difficult to predict the placein the body to perform the surgery to remove the metinel node. In aspecific embodiment of the invention, the lymph node locator is aradioactive substance, such as, e.g. technetium-99m, which may beinjected by a non-surgical procedure, and later imaged by performing alymphoscintigraphy.

Some times the identification of the metinel lymph nodes may involveinjection of lymph node locators by a combination of a non-surgical anda surgical step. As an example of this a radioactive lymph node locator,such as, e.g., technetium-99m may be injected using a needle, i.e.without the need for surgery, and the accumulation of the locator, i.e.identification of the metinel node(s) may be performed using a gammadetector. This gives the surgeon an indication towards where the metinelnodes are located. Later on, when the patient is undergoing surgery tohave the metinel lymph nodes and at least part of metastasis removed, acoloured dye such as, e.g., Patent Blue Dye may be injected.Furthermore, if there is a lapse of more than about 18 to 24 hours afterthe first injection, it might be beneficial to add one or more extrainjections with radioactive tracer dependent on the half-life of theradioactive tracer (usually about 6 hours) in order to identify themetinel nodes during surgery.

After having located the one or more sentinel or metinel lymph nodes byone or the other method, the surgeon will remove these in order toinvestigate whether the sentinel or metinel lymph nodes contain anytumour cells, and in order to obtain a culture of tumour-reactiveT-lymphocytes.

The harvesting of lymphocytes from the one or more sentinel or metinellymph nodes may be performed by homogenizing the sentinel or metinellymph node material in order to obtain single cell suspensions oflymphocytes. The single cell suspensions may then be subjected to invitro expansion in order to obtain tumour-reactive T-lymphocytes.

In Vitro Expansion

The in vitro expansion step iv) of the method according to the inventioncomprises

-   -   i) a first phase of stimulating tumour-reactive CD4+ helper        and/or CD8+ T-lymphocytes with tumour-derived antigen together        with at least one substance having agonistic activity towards        the IL-2 receptor, to promote survival of tumour-reactive CD4+        helper and/or CD8+ T-lymphocytes, and    -   ii) a second phase of activating and promoting growth of        tumour-reactive CD4+ helper and/or CD8+ T-lymphocytes, wherein        the second phase ii) is initiated when the CD25 cell surface        marker (IL-2R marker) is down-regulated on T-lymphocytes.        Phase i)

The purpose of the first phase i) is to obtain a culture comprising asubstantially high ratio of tumour-reactive CD4+ helper and/or CD8+T-lymphocytes. The first phase is to be considered a “nursing phase”where the tumour-reactive T-lymphocytes are brought to survive anddivide. Depending on the source of the T-lymphocytes (starting materialfor the in vitro expansion method), they may have phased relativelyharsh conditions, such as, e.g., suppression and inhibition by factorssecreted by cancer cells.

The starting material for use in the expansion method according to theinvention may be a mixture of lymphocytes obtained from lymph nodesdraining a primary tumour and/or a metastasis, such as, e.g., a sentinelor metinel lymph node. These can be identified during surgery e.g. byinjection of a lymph node locator, such as, e.g., a tracer substance,around or into the tumour or metastasis. The lymph node locator, suchas, e.g., the tracer is transported in the lymph capillaries andaccumulates in the sentinel/metinel node(s), thus identifying the tumouror metastasis draining lymph node(s). The inventors have recently shownthat the first lymph nodes to receive drainage from a tumour are apotential rich source for naturally tumour-reactive CD4+ helper and/orCD8+ T-lymphocytes for in vitro expansion, as such nodes may contain asubstantial amount of T-lymphocytes, that have been sensitized towardstumour-antigens and undergone in vivo expansion in the lymph nodes.

An alternative source of CD4+ helper and/or CD8+ T-lymphocytes may bethe blood of a subject suffering from cancer, such as, e.g., peripheralblood. The subject may be an untreated patient that has had the diseasefor a long time or an already treated patient, wherefrom peripheralT-lymphocytes sensitized towards a tumour may be obtained. Othersuitable sources of CD4+ helper and/or CD8+ T-lymphocytes include bonemarrow, spleen tissue and tumours.

However, in a preferred embodiment of the invention, the startingmaterial is obtained from sentinel or metinel lymph nodes.

The T-lymphocytes to be expanded in culture can be obtained from thesubject to be treated, i.e. the resulting specific tumour-reactiveT-lymphocytes for administering may be autologous. However, theT-lymphocytes can also be obtained from a source other than the subjectto be treated, such as, e.g. another subject suffering from a cancer. Insuch case the recipient and the expanded tumour-reactive T-lymphocytesare preferably immunologically compatible (or the recipient is otherwisemade immuno-tolerant of the expanded tumour-reactive T-lymphocytes).

Depending on the source of the starting material, it will comprise amixture of various lymphocytes, such as, e.g., T-lymphocytes,B-lymphocytes, antigen presenting cells, tumour-reactive T-lymphocytesand non-activated/non-reactive T-lymphocytes. In order to promotesurvival specifically of the tumour-reactive CD4+ helper and CD8+T-lymphocytes, tumour-derived antigen and one or more substances havingagonistic activity towards the IL-2 receptor are added.

As mentioned above the first phase i) is initiated by adding at leastone substance having agonistic activity towards the IL-2 receptor. Thefunction of such substances is to stimulate T-lymphocytes via the IL-2receptor to promote cell division of T-lymphocytes, thereby preventingcell death.

Antigen specific MHC restricted activation of T-lymphocytes promotesclonal expansion of the useful T-lymphocyte population specific for therecognition of tumour cells. On the contrary, unspecific activation of Tlymphocytes will lead to the expansion of T lymphocyte clonesrecognizing irrelevant peptides without any relation to the recognitionof tumour cells, thus the majority of unspecifically expanded Tlymphocytes will not recognize the tumour.

The invention aims to promote specific activation and growth oftumour-reactive CD4+ helper and CD8+ T-lymphocytes. A specificactivation against a certain tumour antigen enables the T-lymphocytes tohave therapeutic effect when administered to a cancer patient with thesame tumour type as the T-lymphocytes are activated against.

Administration of unspecifically activated T-lymphocytes would have noor a very low probability of having therapeutic effect against anycancer, due to the small number of tumour relevant T lymphocytes.

In one embodiment of the invention the substances having agonisticactivity towards the IL-2 receptor are agonists. Examples of suchsubstances include proteins, polypeptides, peptides, antibodies,affibodies, and fragments thereof, fusion proteins, synthetic and/ororganic molecules, such as, e.g., small molecules, and natural ligands.In a preferred embodiment the substance is the natural ligand of theIL-2 receptor, namely IL-2.

If IL-2 is used it is preferentially added in a low dose in order toreduce lymphocyte apoptosis and to increase the population of CD4positive helper tumour-reactive T-lymphocytes. In a specific embodimentof the invention, the low dose of IL-2 is from about 100 IU/ml culturemedium to about 700 IU/ml culture medium, such as, e.g., from about 100IU/ml culture medium to about 600 IU/ml culture medium, from about 100IU/ml culture medium to about 500 IU/ml culture medium, from about 100IU/ml culture medium to about 400 IU/ml culture medium, from about 100IU/ml culture medium to about 300 IU/ml culture medium and from about100 IU/ml culture medium to about 200 IU/ml culture medium. In aspecific embodiment, the amount of IL-2 added is 240 IU/ml.

In case other substances, than IL-2, having agonistic activity towardsthe IL-2 receptor are used the specific doses of these should be suchthat lead to an effect corresponding to the effect obtained by theabove-mentioned doses of IL-2.

A further amount of the at least one substance having agonistic activitytowards the IL-2 receptor may be added regularly throughout phase i),such as, e.g., every 2^(nd), 3^(rd) or 4^(th) day of phase i), in orderto maintain optimal conditions for promoting cell division. By the termevery 2^(nd), 3^(rd) or 4^(th) is intended to mean that at least onesubstance having agonistic activity towards the IL-2 receptor is addedthroughout phase i) every 2^(nd), 3^(rd) or 4^(th) day, starting at the2^(nd), 3^(rd) or 4^(th) day after the first addition of the at leastone substances having agonistic activity towards the IL-2 receptor, i.e.after initiating phase i).

In one embodiment the substance to be added regularly throughout phasei) is an agonist of IL-2. In a preferred embodiment the substance isIL-2.

The further dose of substances having agonistic activity towards theIL-2 receptor, such as, e.g., IL-2, to be added regularly, such as, e.g.every 2^(nd), 3^(rd), or 4^(th) day lies within the ranges mentionedabove.

A further important step in the first phase i) of expansion is theaddition of tumour-derived antigen in order to promote cell division ofT-lymphocytes expressing T lymphocyte receptors recognizing tumourantigens, i.e. tumour-reactive T-lymphocytes.

The optimal point of time to add the tumour-antigen is depending on thesource of lymphocytes. When the lymphocytes originates from lymph nodes,such as, e.g., sentinel lymph nodes, or from tumours, the lymphocytesmay have been subjected to close proximity and immuno-suppression bytumour cells, and need incubation with a substance having agonisticactivity towards the IL-2 receptor, such as, e.g., IL-2 for some days inorder to promote the ability of the T-lymphocytes to respond withproliferation upon tumour antigen presentation. Accordingly, in suchcase the tumour-derived antigen is preferentially added from day 2 toand including day 5 of the first phase i), such as, e.g., on day 2, onday 3, on day 4 or on day 5.

If the lymphocytes originate from blood, the tumour-derived antigen maybe added already when the first phase i) is initiated, i.e. togetherwith the substance having agonistic activity towards the IL-2 receptor,as the T-lymphocytes have not been subjected to the above-mentionedimmuno-suppression by tumour cells. Accordingly, when blood is used, thetumour-derived antigen is added essentially at the same time as whenphase i) is initiated or at the most up to 2 days thereafter.

The tumour-derived antigen, such as, e.g., a tumour homogenate, islikely to be endocytosed and processed by antigen presenting cellspresent in the starting material, such as, e.g., B-lymphocytes,dendritic cells and macrophages. In most cases the tumour-derivedantigen will be presented by class II MCH molecules leading to celldivision of CD4⁺ helper tumour-reactive T-lymphocytes. However, by crosspresentation antigens taken up by endocytosis may be processed andpresented in the class I pocket resulting in activation of CD8⁺ Tlymphocytes. As stated above, one of the objects of the expansion methodis to in some respect imitate the natural pathway of the patients ownimmune system, and to a certain degree let the components of thepatients immune system determine whether CD4⁺ or CD8⁺ lymphocytes aregenerated, depending on whether antigen is presented by MCHI or MCHII.In most cases, the antigens will be presented by the class II MCHmolecule leading to generation of CD4⁺ T-lymphocytes, however, in somecases CD8⁺ T-lymphocytes are generated.

In a specific aspect of the invention, at the end of phase i) the sum ofCD4⁺ and CD8⁺ T-lymphocytes isolated from a small intestine cancercomprise at least 70% of the cell population, such as at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% or 100%. In anotherspecific aspect of the invention, at the end of phase i) CD4⁺T-lymphocytes isolated from a small intestine cancer comprise at least50% of the cell population, such as at least 60%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95%. In yetanother specific aspect of the invention, at the end of phase i) CD8+T-lymphocytes isolated from a small intestine cancer comprise at least1% of the cell population, such as at least 5%, at least 10%, at least15%, at least 20%, or at least 25%. In a further specific aspect of theinvention, at the end of phase i) the number of CD4+ and/or CD8+T-lymphocytes isolated from a small intestine cancer has increased by afactor 1.5, such as 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, or 20. In anotherfurther specific aspect of the invention, at the end of phase i) theratio of CD4+ to CD8+ T-lymphocytes isolated from a small intestinecancer has increased by a factor 1, such as 1.25, 1.5, 1.75, 2, 3, or 4as compared to the beginning of the expansion.

In a specific aspect of the invention, at the end of phase i) the sum ofCD4+ and CD8+ T-lymphocytes isolated from a gallbladder cancer compriseat least 70% of the cell population, such as at least 75%, at least 80%,at least 85%, at least 90%, at least 95% or 100%. In another specificaspect of the invention, at the end of phase ii) CD4+ T-lymphocytesisolated from a gallbladder cancer comprise at least 70% of the cellpopulation, such as at least 75%, at least 80%, at least 85%, at least90%, or at least 95%. In yet another specific aspect of the invention,at the end of phase ii) CD8+ T-lymphocytes isolated from a gallbladdercancer comprise at least 1% of the cell population, such as at least 5%,at least 10%, at least 15%, at least 20%, or at least 25%.

Phase ii)

The purpose of the second phase ii) is to activate and expand thetumour-reactive CD4+ helper and/or CD8+ T-lymphocytes obtained by phasei) and to obtain a specific sub-population of tumour-reactive CD4+helper and/or CD8+ T-lymphocytes by directing them into a desiredpathway.

The present inventors have found, that one way of determining theoptimal point in time to initiate phase ii) is by monitoring theexpression of the CD25 cell surface marker on the T-lymphocytes, inorder to determine specifically when the T-lymphocytes are susceptibleto re-stimulation. The present inventors have found that the secondphase ii) should preferably be initiated when the expression of CD25 onT-lymphocytes is down-regulated. CD25 is an activation marker,indicating that the lymphocytes have received an activating signal. Ifthe second phase is initiated when the expression of CD25 on theT-lymphocytes is high, meaning that the lymphocytes have alreadyreceived a signal, cell death would occur.

The down-regulation of CD25 is defined as that a substantial part of theT-lymphocyte population express very few or essentially none CD25markers. In a preferred embodiment the down-regulation of CD25 isdefined as that less than 5% of the T-lymphocyte population expressesCD25, i.e. 95% or more of the T-lymphocytes in the culture do notexpress CD25 at all. The 5% or less of the T-lymphocytes expressing CD25are most likely regulatory CD4+ T-lymphocytes, which have a highpermanent expression of CD25. In addition, the T-lymphocyte populationshould preferably express very few or essentially none Foxp3 markers,which are specific markers of regulatory T-lymphocytes. In a preferredembodiment the down-regulation of Foxp3 is defined as that less than 5%of the T-lymphocyte population expresses Foxp3, i.e. 95% or more of theT-lymphocytes in the culture do not express Foxp3 at all.

Besides CD25, there are also other markers, the expression of which isrelevant to monitor in order to determine the optimal point in time toinitiate the second phase. Examples of such markers are the earlyactivation marker CD69, and MCHII, which is an activation markerforT-lymphocytes. As the expression of CD69 and MCHII indicates that the“activation program” of the T-lymphocytes is already turned on, meaningthat the cells are not able to respond to additional stimuli, both ofthese markers should preferably be down-regulated before the secondphase is initiated. The term down regulation may be defined as that lessthan 5-10% of the T-lymphocyte population expresses CD69 and/or MCHII.

In another embodiment of the present invention, anti-CD4 antibodies areused to separate T helper cells from possible tumour cells in theculture in the expansion in phase ii) of the expansion method.

In a further or yet another embodiment of the present invention,products such as Dynabeads® with anti-CD3 and anti-CD28 antibodies areused to promote the expansion in phase ii) of the expansion method. Useof Dynabeads® CD3/CD28 will provide lymphocytes with activation signalsand could also be used for separation from possible tumour cells in theculture. Dynabeads® CD3/CD28 will bind to T lymphocytes expanded antigenspecifically during phase i), where these cells now can be enrichedmagnetically. Since the initial antigen specific activation hasinitiated and led to clonal T lymphocyte expansion the Dynabeads®CD3/CD28 restimulation will further promote clonal expansion since phasei) does not support activation of unspecific T lymphocyte clones.

Even though the exact starting point of phase ii) will vary depending onwhen the lymphocytes has acquired the preferred expression of specificmarkers, the second phase ii) is most often initiated from day 17 to andincluding day 23 of the first phase i), such as, e.g. on day 17, on day18, on day 19, on day 20, on day 21, on day 22 or on day 23. In otherwords, the point in time, where the lymphocytes expresses the preferredamount and combination of markers, is most often seen as being from day17 to day 23 of the first phase i).

The expansion of the T-lymphocytes, i.e. phase i) and ii) will mostoften take place in a suitable culture medium. Preferably a serum-freemedium or autologous serum is used in order to avoid the risk oftransmitting diseases to the patient. Examples of suitable standardmedia include AIMV medium, RPMI 1640, DMEM and MEM. However, other mediamay also be used, comprising a suitable blend of amino acids, steroids,vitamins, growth factors, cytokines and minerals.

During the two phases of the expansion, the cells may be split intoseveral culture vessels in order to maintain a suitable cell density inthe cultures. The density of the T-lymphocytes in the expansion phasesshould preferably be from about 3 to about 6 million cells/ml of culturemedium.

During expansion an exchange of culture medium with fresh medium, astep, which is denominated conditioning of the medium, may also beneeded. The point of time to split cultures and to condition the mediummay be determined based on the morphology of the cells and the cellculture density (which should not exceed about 6 million cells/ml), orthe medium may contain a suitable indicator, such as, e.g., a phenolindicator. In case an indicator is included in the medium, the point oftime to split cultures or condition medium may be based on the color ofthe medium. If a phenol red indicator is used, the cells should be splitor conditioned, when the medium turns yellow, indicating that the pH ofthe culture is turning acidic. A suitable schedule for conditioning themedium used in the present invention may be to exchange from ¼ to ½,such as, e.g., ⅓ of the medium every 3-9 days, such as, e.g. once aweek.

Except for the specific conditions mentioned herein, for otherparameters standard conditions for growth of lymphocyte cultures will beused, such as, e.g. a temperature of 37° C. and 5% CO₂.

As mentioned above, the second phase ii) is initiated by the addition oftumour-derived antigen as defined above to the T-lymphocytes foractivating the tumour-reactive CD25-negative T-lymphocytes, in order topromote clonal expansion of tumour-reactive T-lymphocytes.

In a specific embodiment of the invention antigen presenting cells(APCs) are added to the T-lymphocytes together with the tumour-derivedantigen. Antigen presenting cells (APCs) include leukocytes such as,e.g., monocytes, macrophages and lymphocytes, such as, e.g., B cells.These diverse cell types have in common the ability to present antigenin a form that is recognized by specific T lymphocyte receptors. Theleukocyte preparation is isolated from, for example, blood, lymph fluid,bone marrow, lymphatic organ tissue or tissue culture fluid obtainedfrom the patient to be treated. In a preferred embodiment the APCs cellsare irradiated peripheral blood leucocytes containing antigen-presentingB-cells and/or monocytes. The amount of APCs added lies within the rangeof from about 0.5 million APCs/ml lymphocyte culture to about 5 millionAPC/ml lymphocyte culture, such as, e.g., from about 1 million APCs/mllymphocyte culture to about 4 million APC/ml lymphocyte culture, fromabout 1 million APCs/ml lymphocyte culture to about 3 million APC/mllymphocyte culture, or from about 1 million APCs/ml lymphocyte cultureto about 2 million APC/ml lymphocyte culture.

Besides the addition of tumour-derived antigen to the T-lymphocytes inorder to promote clonal expansion of tumour-reactive T-lymphocytes, thesecond phase ii) comprises the addition of specific components thefunction of which are to direct the expansion of the tumour-reactiveT-lymphocytes towards the desired sub-population.

As mentioned above, the present invention provides a method for thegeneration of tumour-reactive CD4+ helper T-lymphocytes. CD4+ helperT-lymphocytes recognizes and binds tumour antigen when the antigen isassociated with a major histocompatibility complex class II molecule.Activated CD4+ helper T lymphocytes secrete cytokines, proteins and/orpeptides that stimulate other cells of the immune system, such as otherlymphocytes. The most common cytokine secreted is interleukin-2 (IL-2),which is a potent T lymphocyte growth factor. Activated, proliferatingCD4+ helper T-lymphocytes can differentiate into two major subtypes ofcells, Th1 and Th2 cells, which are defined on the basis of specificcytokines produced. Th1 cells produce interferon-gamma and interleukin12 (IL-12), while Th2 cells produce interleukin-4, interleukin-5 andinterleukin-13. Th1 T-lymphocytes are believed to promote activation ofcytotoxic T lymphocytes (Tc), NK cells, macrophages, and monocytes, allof which can attack cancer cells and generally defend against tumours.

T-helper (CD4+) lymphocytes of type Th1 and Th2 can differentiate intomemory cells and effector cells. Memory T-helper (CD4+) lymphocytes arespecific to the antigen they first encountered and can be called uponduring a secondary immune response, calling forth a more rapid andlarger response to the tumour-antigens. There is evidence in humans thatlymphocytes survive at least 20 years; perhaps for life. Effector CD4+T-lymphocytes are active cells producing cytokines and INF-gamma. For aneffective treatment of cancer, administration of tumour-reactiveT-lymphocytes of the Th1 type is especially beneficial, as this type isbelieved to promote activation of cytotoxic T lymphocytes (Tc), NKcells, macrophages, and monocytes, all of which can attack cancer cellsand generally defend against tumours. I.e. in a specific embodiment theinvention relates to a method for generating tumour-reactive CD4+ helperT-lymphocytes, and in a further embodiment, the percentage ofT-lymphocytes of the Th2 type generated by the present method is 30% orless, such as, e.g., 25% or less, 20% or less, 15% or less, 10% or less,5% or less or 0%, i.e. at least 70% of the tumour-reactive CD4+T-lymphocytes are of the Th1 type, such as, e.g. at least 75%, at least80%, at least 85%, at least 90%, at least 95% or 100%.

Accordingly, the second phase may comprise the addition of a substancecapable of up-regulating IL-12R on the T-lymphocytes. Up regulation ofthe IL-12R will increase the readiness of the T cell to receive andoptimize the IL-12 cytokine activation resulting in maximal STAT-4signalling and thus skewing the lymphocytes towards Th1 cells and IFN-γproduction.

The substance(s) capable of up-regulating IL-12R on the T-lymphocytesmay be substance(s) having agonistic activity towards an interferonreceptor. In one embodiment of the invention the substances havingagonistic activity towards the interferon receptor are agonists.Examples of such substances include proteins, polypeptides, peptides,antibodies, affibodies, and fragments thereof, fusion proteins,synthetic and/or organic molecules, such as, e.g., small molecules, andnatural ligands. In a specific embodiment the substance is the naturalligand of the interferon receptor, namely an interferon, such asinterferon-α.

The optimal point of time to add the substance(s) capable ofup-regulating IL-12R on the T-lymphocytes, such as, e.g. a substancehaving agonistic activity towards an interferon receptor may bedetermined by measuring the level of IL-12 in the culture medium. Thesubstance(s) should preferably be added when the level of IL-12 is atleast 1 fold, such as, e.g., at least 2, at least 3 fold, at least 4fold, or at least 5 fold increased as compared to the level of IL-12 onday 1 of phase ii). In most cases, such an increase in the level ofIL-12 will be seen from day 2 to and including day 4 after initiatingthe second phase ii), such as, e.g. on day 2, on day 3 or on day 4.

In order to substantially avoid the generation of tumour-reactiveT-lymphocytes of the Th2 type, the second phase may further comprise theaddition of one or more substances capable of antagonizing developmentof Th2 type T-lymphocytes. Examples of such substances are substancescapable of neutralizing the interleukins IL-4, IL-5, IL-10, and/orTGF-beta (the latter not being an interleukin) all four of which arerequired for the establishment of the Th2 cytokine profile and for downregulation of Th1 cytokine production.

Examples of such substances include proteins, polypeptides, peptides,soluble receptors, antibodies, affibodies, and fragments thereof, fusionproteins, synthetic and/or organic molecules, such as, e.g., smallmolecules, and natural ligands. In a specific embodiment the substancesare selected from antibodies that binds to the interleukins, therebyneutralizing them, such as, e.g. anti IL-4 antibody, anti IL-5 antibodyand/or anti IL-10 antibody, together with soluble receptors (such as,e.g. TGF-beta receptor I and II) and binding proteins for TGF-beta (suchas, e.g. LAP and/or LTBP).

The one or more substances capable of antagonizing development of Th2type T-lymphocytes, such as, e.g., one or more substances capable ofneutralizing IL-4, IL-5, IL-10 and/or TGF-beta may be added on day 1 ofthe second phase ii). However, as antibodies are expensive, the additionof antibodies can also be performed in a subsequent step after additionof the substance capable of up-regulating IL-12R on the T-lymphocytes,such as, e.g., one day, two days or three days after addition of thesubstance capable of up-regulating IL-12R on the T-lymphocytes.

The neutralizing substances should be added in an amount sufficient toneutralize the interleukins, such as, e.g., in a 10-100 fold (molar)excess of the amount of interleukin to be neutralized. When usingantibodies, a final concentration of from about 2 to about 4 ng/mlculture medium will normally be needed. For other types of neutralizingsubstances, a final concentration, giving the same effect as theconcentration mentioned for antibodies, should be used.

In order to maintain the suppression of the development of Th2 typeT-lymphocytes a further amount of the one or more substance capable ofantagonizing development of Th2 type T-lymphocytes, such as, e.g., oneor more substance capable of neutralizing IL-4, IL-5, IL-10 and/orTGF-beta may be added regularly throughout phase ii), such as, e.g.every 2^(nd), 3^(rd) or 4^(th) day of phase ii). It is to be understoodthat by the term every 2^(nd), 3^(rd) or 4^(th) is intended to mean thatat least one substance capable of antagonizing development of Th2 typeT-lymphocytes is added throughout phase i) every 2^(nd), 3^(rd) or4^(th) day, starting at the 2^(nd), 3^(rd) or 4^(th) day after the firstaddition of the at least one substance capable of antagonizingdevelopment of Th2 type T-lymphocytes.

Furthermore, as for phase i) a further amount of a substance havingagonistic activity towards the IL-2 receptor, such as, e.g., an agonistmay be added regularly throughout phase ii) such as, e.g., every 2^(nd)to 4^(th) day of phase ii), i.e. on the 2^(nd), 3^(rd) or 4^(th) day inorder to maintain optimal conditions promoting cell division. The doseof the substance to be added regularly lies within the optimal rangesmentioned under phase i) for addition of substances having agonisticactivity towards the IL-2 receptor, such as, e.g., IL-2.

In order to favor the generation of Th1 type tumour-reactiveT-lymphocytes, the second phase ii) may comprise adding one or moresubstances promoting the development of Th1 type T-lymphocytes. Examplesof such substances are substances having agonistic activity towards theIL-7, IL-12, IL-15 and/or IL-21 receptor. More specific, the substancesmay be agonists for the IL-7, IL-12, IL-15 and/or IL-21 receptor.Examples of such agonists include proteins, polypeptides, peptides,antibodies, affibodies, and fragments thereof, fusion proteins,synthetic and/or organic molecules, such as, e.g., small molecules, andnatural ligands. In a specific embodiment the substances are the naturalligands of the IL-7, IL-12, IL-15 and/or IL-21 receptor, respectively,such as IL-7, IL-12, IL-15 and/or IL-21.

The effect of IL-12 is activating the IFN-gamma inducing STAT pathway bystimulating the IL-12R thereby promoting activation of Th1 lymphocytes.The function of IL-21 is to enhance proliferation, activation anddevelopment towards a Th1 type of T-lymphocytes.

Both IL-7 and IL-15 work by promoting homeostatic expansion of theT-lymphocytes, enhancing the enumeration of activated Th1 programmedT-lymphocytes.

The optimal point of time to add one or more substances promotingdevelopment of Th1 type T-lymphocytes is when the T-lymphocytes aresusceptible to modification. If the substances are added when theT-lymphocytes are not susceptible to modification, the addition willhave no effect, i.e. the development of Th1 type T-lymphocytes will notbe favoured. In order to determine the optimal point in time for addingsubstances promoting development of Th1 type T-lymphocytes, such as,e.g., substances having agonistic activity towards the IL-7, IL-12,IL-15 and/or IL-21 receptor, the production of INF-γ by theT-lymphocytes, may be monitored. In a preferred embodiment, the one ormore substances promoting the development of Th1 type T-lymphocytes,such as, e.g., substances having agonistic activity towards the IL-7,IL-12, IL-15 and/or IL-21 receptor should be added when the level ofIFN-gamma is increased as compared to the level of IFN-gamma oninitiation of second phase ii).

In a specific embodiment, the increase in IFN-gamma level may bedetermined as at least a 1 fold increase in IFN-gamma level, such as,e.g., at least a 2 fold, at least a 3 fold, at least a 4 fold increaseas compared to the level of IFN-gamma on initiation of the second phaseii). Often will such an increase can be correlated to that the contentIFN-gamma in the culture medium should be at least 100 picogram/mlculture medium, such as, e.g. at least 150 picogram/ml culture medium,at least 200 picogram/ml culture medium or at least 250 picogram/mlculture medium.

When determining the optimal point in time to add substances promotingdevelopment of Th1 type T-lymphocytes, such as, e.g., substances havingagonistic activity towards the IL-7, IL-12, IL-15 and/or IL-21 receptor,one may further look at the expression of the activation markers CD25and CD69 on CD4+ T-lymphocytes, which markers should preferentially beup-regulated. By up-regulation is understood that at least about 40% toabout 60% or more of the CD4+ T-lymphocytes should express CD25 and CD69as compared to the expression of CD25 and CD69 on T-lymphocytes on day 1of phase ii), showing that the T-lymphocytes have received an activatingsignal.

Normally the optimal point of time for adding the substances promotingdevelopment of Th1 type T-lymphocytes will fall subsequent to the stepsof adding the substances capable of up-regulating IL-12R on theT-lymphocytes and the substances capable of antagonizing development ofTh2 type T-lymphocytes. More specific the optimal point in time to addthe substances promoting development of Th1 type T-lymphocytes will fallbetween day 5 to day 8 after initiating the second phase ii), such as,on day 5, day 6, day 7 or day 8.

In case IL-7, IL-12, IL-15 and/or IL-21 are added the concentration ofeach of these substances in the culture medium should lie within therange from about 150 IU/ml culture medium to about 300 IU/ml culturemedium, such as, e.g. 250 IU/ml culture medium. When other substancesthan the specific ones mentioned is used, they should be added to theculture in final concentration, which leads to the same effect as theaddition of IL-7, IL-12, IL-15 and/or IL-21 within the specific rangesmentioned will give.

As mentioned above, the present method is preferentially used for theexpansion of T-lymphocytes in order to achieve CD4+ tumour-reactiveT-lymphocytes of the Th1 type. One further aspect of the invention isthat by using the method described herein for expanding tumour-reactiveT-lymphocytes, a relatively high amount of T-lymphocytes of the memorytype will be obtained. In treating cancer it is of course important thatthe patient to be treated receive a high amount of effectortumour-reactive CD4+ T-lymphocytes, as these—as mentioned above—promoteactivation of cytotoxic T lymphocytes (Tc), NK cells, macrophages, andmonocytes, all of which can attack cancer cells and generally defendagainst tumours.

However, by at the same time administering a substantial amount ofmemory tumour-reactive CD4+ T-lymphocytes, the patient achieve up tolife long protection towards recurrence of the tumour or metastasis ofthe primary tumour.

Accordingly, the present invention relates to a method for thepreparation of memory T-lymphocytes. Normally, when a culture oftumour-reactive T-lymphocytes are expanded according to the presentinvention from about 35% to about 90% of tumour-reactive T-lymphocytesof the memory type, such as, e.g. from about 40% to about 90%, fromabout 50% to about 80% or from about 60% to about 70%, will be obtained.The present inventors speculates that the fact that the lymphocytes inphase i) are allowed to regenerated before tumour antigen is added,together with the relatively slow expansion phase leads to formation ofa high ratio of memory lymphocytes to effector lymphocytes.

As mentioned above the expression of the cell surface activation markersCD25 and CD69 on the T-lymphocytes may be used for determining when toinitiate important steps of the present method, such as, e.g., when toinitiate the second phase ii). Accoridngly, it may be beneficial tocontinuously monitor the expression of CD25 and CD69 throughout phase i)and phase ii), such as, e.g., every 2^(nd), every 3^(rd) or every 4^(th)day.

As one of the purposes of the present method is to obtain a high numberof specific CD4+ tumour-reactive T-lymphocytes, which may be used foradministering to a patient, the tumour-reactive T-lymphocytes may beharvested at some point, leading to the termination of the expansionstep. The optimal point of time to harvest the tumour-reactiveT-lymphocytes is when the expression of CD25 on the T-lymphocytes isdown-regulated, where the down-regulation is defined as that 5% or lessof the CD4+ T-lymphocyte population expresses CD25. The optimal point intime to harvest may also be determined based on measurement of theamount of IFN-gamma produced. The IFN-gamma production should be atleast 2 fold increased, such as, e.g., at least 3 fold, at least 4 foldor at lest 5 fold increased as compared to initial IFN-gamma production,which normally correspond to a level of IFN-gamma of at least 100 pg/mlof culture medium.

Normally, this event will occur from day 10 to and including day 14after initiating the second phase ii), i.e. normally the cells will beharvested from day 10 to and including day 14 after initiating thesecond phase ii).

Accordingly, the entire process for expansion of tumour-reactiveT-lymphocytes according to the invention may in general take from about25 days to and including about 45 days, such as, e.g. from about 26 daysto and including about 44 days, from about 27 days to and including 43days, from about 27 days, to and including 42 days, from about 27 daysto and including 41 days, and from about 27 days to and including about40 days.

Instead of harvesting the tumour-reactive T-lymphocytes when the CD25marker is down regulated, they may be subjected to one or moreadditional rounds of phase ii). This could be beneficial to do if theamount of tumour-reactive T-lymphocytes obtained by the expressionmethod is not considered an effective amount to be administered to apatient suffering from cancer, or if the patient is in a chemo-therapytreatment regimen, where it may be considered beneficial to postpone theadministration of T-lymphocytes until the chemo-therapy treatment isfinished. In order to determine whether the tumour-reactiveT-lymphocytes should be subjected to one or more additional rounds ofphase ii) one may look at the level of IFN-gamma produced, and/or thetotal number of tumour-reactive T-lymphocytes obtained and/or theexpression of CD25. In the case the IFN-γ levels is 30 pg/ml culturemedium or less, such as, e.g. 20 pg/ml culture medium or less, and/orthe total number of T cells are unsatisfactory, additional rounds ofphase ii) may be initiated beginning when the majority of T cells areCD25 negative (i.e. less than 5% of the T-lymphocytes population expressCD25) and thereby susceptible to restimulation.

After harvest the tumour-reactive T-lymphocytes may be purified by anyconventional means, such as, e.g. by using density gradient, such as,e.g., a Ficoll medium. A portion of the tumour-reactive T-lymphocytesmay be stored by freezing in a suitable freezing medium after harvestingand purifying the tumour-reactive T-lymphocytes.

In a specific aspect of the invention, at the end of phase ii) the sumof CD4+ and CD8+ T-lymphocytes isolated from a small intestine cancercomprise at least 70% of the cell population, such as at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% or 100%. In anotherspecific aspect of the invention, at the end of phase ii) CD4+T-lymphocytes isolated from a small intestine cancer comprise at least50% of the cell population, such as at least 60%, at least 70%, at least80%, at least 90%, or at least 95%. In yet another specific aspect ofthe invention, at the end of phase ii) CD8+ T-lymphocytes isolated froma small intestine cancer comprise at least 5% of the cell population,such as at least 10%, at least 15%, at least 20%, at least 25%, at least30%, or at least 35%.

In a specific aspect of the invention, at the end of phase ii) the sumof CD4+ and CD8+ T-lymphocytes isolated from a mesothelioma comprise atleast 70% of the cell population, such as at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% or 100%. In another specificaspect of the invention, at the end of phase ii) CD4+ T-lymphocytesisolated from a mesothelioma comprise at least 50% of the cellpopulation, such as at least 60%, at least 70%, at least 80%, at least90%, or at least 95%. In yet another specific aspect of the invention,at the end of phase ii) CD8+ T-lymphocytes isolated from a mesotheliomacomprise at least 5% of the cell population, such as at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, or atleast 35%.

Method of Treatment

The tumour-reactive T-lymphocytes obtained by an improved expansionmethod as described above may be used in a method for treating a subjectsuffering from a disease of neoplastic origin or for effecting tumourregression in a subject having a tumour, the method comprisingadministering to the subject in need thereof an effective amount oftumour-reactive T-lymphocytes according to the invention.

The method described herein may be used for treatment of any solidneoplasm of epithelial, mesenchymal or embryological origin in anyanatomical location, such as e.g., for epethilal neoplasms e.g.carcinomas in the breast, colon, pancreas, bladder, small intestines,prostate, cervix, vulva, ovaries; for mesenchymal neoplasms e.g.sarcomas in the joints, bones, muscles and tendons and somehaematological such as lymphomas; for embryological neoplasms, e.g.teratomas.

In a specific embodiment the present invention provides a method for usefor the treatment of a neoplastic disease selected from gall bladdercancer, hepato cellular cancer, ovarian cancer, small intestine cancer,lung cancer, mesothelioma, breast cancer, kidney cancer, pancreascancer, prostate cancer, carcinoid cancer, leiomyosarcoma, or metastasisthereof.

The tumour-reactive T-lymphocytes obtained by an improved expansionmethod are used herein for treating patients suffering from a gallbladder cancer, hepato cellular cancer, ovarian cancer, small intestinecancer, lung cancer, mesothelioma, breast cancer, kidney cancer,pancreas cancer, prostate cancer, carcinoid cancer, leiomyosarcoma, ormetastasis thereof.

The definition of an effective amount of tumour-reactive T-lymphocytesis depending on the specific type of lymphocytes, the ratio of memory toeffector T-lymphocytes and on the severity of the disease. However, inaverage a minimum of at least 10 million, such as, e.g. at least 20million, at least 30 million, at least 40 million, at least 50 million,at least 60 million, at least 70 million or at least 80 milliontumour-reactive T-lymphocytes may be administered. The present inventorshave not identified any upper limit with respect to the amount oftumour-reactive T-lymphocytes to be administered in a single dose.

In a preferred embodiment the tumour-reactive T-lymphocytes foradministration comprises a combination of effector T-lymphocytes andmemory T-lymphocytes. More specific the amount of tumour-reactiveT-lymphocytes of the memory type may be from about 35% to about 90%,such as, e.g. from about 40% to about 90%, from about 50% to about 80%or from about 60% to about 70%, and a percentage of effectorT-lymphocytes from about 10% to about 65%, such as, e.g., from about 20%to about 50% or from about 30% to about 40%.

The tumour-reactive T-lymphocytes may be formulated as a pharmaceuticalcomposition suitable for parenteral administration to the patient suchas, e.g., intravenous, intraarterial, intrathecal, or intraperitonaladministration.

When the tumour-reactive T-lymphocytes are administered parenterally,they may be formulated in an isotonic medium, i.e. in a medium havingthe same tonicity as blood, and comprising one or more substancespreventing aggregation of the cells. A specific example of a suitablemedium is a 0.9% NaCl solution comprising up to 3% human serum albuminsuch as, e.g. up to 2% human serum albumin or up to 1% human serumalbumin. For intravenously administration the concentration oftumour-reactive T-lymphocytes in the composition to be administerednormally lies within the range from about 0.5 million lymphocytes/mlmedium to about 4 million lymphocytes/ml medium, such as, e.g., fromabout 0.5 million lymphocytes/ml medium to about 3 millionlymphocytes/ml medium, from about 0.5 million lymphocytes/ml medium toabout 2 million lymphocytes/ml medium or from about 1 millionlymphocytes/ml medium to about 2 million lymphocytes/ml medium.

The composition comprising tumour-reactive T-lymphocytes may beadministered as a single dose or multiple doses. It may be infused over1 to 2 hours.

The treatment method may be performed once or repeated depending on theseverity of the disease. Furthermore, the treatment may be reiteratedupon recurrence of the disease.

The treatment according to the present invention may be supplementedwith any other relevant treatment for cancer. Such supplementaltreatment may be given before, at the same time or after theadministration of the lymphocytes and it may be given at frequenciesnormally used for such treatments. A suitable example of supplementaltreatment is chemotherapy and the like.

Kits

The invention further relates to kits for use in a method according tothe invention, the kit comprising a medium for cultivation ofT-lymphocytes. The medium may be any suitable serum-free medium, suchas, e.g., AIMV, RPMI 1640, DMEM or MEM.

The kit may further comprise one or more substances for stimulating,activating and directing tumour-reactive T-lymphocytes. Examples of suchsubstances may be tumour-derived antigen, substances having agonisticactivity towards the IL-2 receptor, substances capable of up-regulatingIL-12R on the T-lymphocytes, substances capable of antagonizingdevelopment of Th2 type T-lymphocytes and/or substances promoting thedevelopment of Th1 type T-lymphocytes.

More specific, such substances may be IL-2, interferon-alpha, anti-IL-4antibody, anti-IL-5 antibody, anti-IL-10 antibody, IL-7, IL-12, IL-15and/or IL-21.

The kit may also comprise a pharmaceutical composition suitable forintravenous administration. The pharmaceutical composition may be mixedwith the population of tumour-reactive T-lymphocytes beforeadministration.

The invention also relates to a kit for identification of sentinel ormetinel lymph nodes, the kit comprising one or more syringes and a lymphnode locator. In one embodiment, the syringes are prefilled with a lymphnode locator.

The kits may also comprise instructions for use, such as, e.g.instructions in the form of computer software.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates that the sentinel node is the natural primary sitefor the presentation and activation of T cell reactivity towards tumourantigen.

FIG. 2 shows that initially sentinel node lymphocytes are activated withtumour antigen and low dose IL-2 resulting in activation and expressionof the activation marker CD25 (Top panel). The end of phase I activationphase is defined by the decreased number of CD4⁺ T cells expressing CD25(Bottom panel). When less than 5% of the CD4⁺ T cells express CD25 phaseII is initiated with restimulation with antigen.

FIG. 3 illustrates that Phase I and Phase II activation results inexpansion and enrichment of CD4⁺ T helper cells.

FIG. 4 illustrates that in Phase I the majority of cells are naïveCD62L+ cells or activated CD69+CD62L+ cells. After Phase II the majorityof the cells are CD62L- and are composed of memory and effector CD4+ Thelper cells. CD62L-T cells are not expressing the preferred lymph nodehoming molecule, thus they are seeking inflammatory areas innon-lymphatic organs.

FIG. 5 shows primary cells stimulated in Phase I from the tumour (Tumourinfiltrating lymphocytes), sentinel nodes (SN) and an irrelevant lymphnode (LN) results in no little IFN-γ production.

FIG. 6 illustrates that after expansion after phase ii) there is a dosedependent increase in antigen dependent IFN-γ production.

FIG. 7 illustrates that the expansion and activation protocol promotesthe expansion of antigen specific T cell clones as investigated by theselective enrichment of TCR VP expression.

FIG. 8 A-D are CT scans of patient # 5. After transfusion oftumour-reactive lymphocytes the patient had total regress of livermetastases located in both lobes (which had been declared incurable byliver surgery), normalisation of CEA levels, disappearance of ascitesand was physically well fit, exercising regularly.

FIG. 9 A-F are CT scans of patient # 10. After transfusion the patienthad regress of liver metastases and ascitic fluid. He was in fairly goodhealth and further imaging showed stable disease.

FIG. 10 A-H are CT scans of patient # 12. Three months after transfusionhe had regress of metastases in the liver and lungs with almost anormalised CEA level at 5.9 (Normal<4.0), disappearance of ascites andhe appears clinically healthy.

FIG. 11 shows T lymphocytes gated for the expression of CD4⁺ which werestained for the expression of CD25 and the transcription factor FoxP3 atthe beginning (A) and at the end (B) of an expansion. Initially (panelA), 4.8% of CD4+ T lymphocytes expressed FoxP3 and high levels of CD25,thus identified as Treg. At the end of the expansion a very small numberof Tregs were present 0.3% (panel B).

FIGS. 12 and 13 show results of analysis of Vbeta profiles of CD4 Tcells from patient with mesothelioma (described in example 5). Analysiswas performed on day 2 (Before) and day 24 (After) of the expansions.Clonal expansion of CD4 T cells belonging to Vbeta groups 4 and 14 canbe seen in Culture 1. A similar general pattern of clonal expansion canbe seen in both cultures.

EXAMPLES Example 1 Expansion of Tumour-Reactive T-Lymphocytes

Identification of sentinel nodes was done peroperatively using thesentinel node technique. Briefly, 1 ml of Patent blue dye was injected(Guerbet, Paris) and distributed superficially in the serosa around thetumour. Within five to ten minutes, one to three mesenteric lymph nodeswere coloured blue, these sentinel nodes were marked with sutures andremoved (see FIG. 1). One non-sentinel mesenteric lymph node, distantfrom the tumour, was also identified and removed as a control.

The sentinel- and non-sentinel lymph nodes were cut in half and 1 mmthick slices were taken from the center and the periphery. The rest ofthe lymph nodes were sent for histopathological examination according toroutine procedure. A part of the tumour, including a sample of theinvasive margin, was also removed for research purposes.

Cell Culture

Phase 1, Initial Activation

The sentinel node material was kept on ice and immediately taken care ofusing AIM V® Media (Invitrogen) at all times. Single cell suspensions ofsentinel node lymphocytes were obtained through gentle homogenisation ina loose fit glass homogenisator, and following homogenisation cells werewashed twice in medium. The sentinel node lymphocytes were put in cellculture flasks at 4 million cells/ml and interleukin-2 (IL-2)(Proleukin®, Chiron) was added to a concentration of 240 IU/ml medium.

Autologous tumour extract was prepared by homogenisation with an UltraTurrax in 5 volumes (w/v) 2×PBS followed by denaturation for 5 minutesat 97° C. Three to four days after initiation of the cell cultureautologous tumour extract was added at a concentration of 1/100. Forlong-term culture the cells were kept in a cell incubator at 37° C. and5% CO₂ and 240 IU IL-2/mL media added every 3-4 days.

Phase II, Activation and Expansion

After 18-22 days the cell cultures were monitored for the expression ofCD25. When the number of CD25 expressing cells was diminished below 5%the cells were restimulated in Phase II (FIG. 2) by the addition ofautologus tumour extract at a concentration of 1/100. For efficientantigen presentation autologous PBMC were collected using Ficoll-PaquePLUS (Amersham Biosciences, GE Healthcare), radiated with 2500 rad andadded to the cell cultures. Three days after restimulation interferon-α(Introna) in conc. 100-500 IU/ml and anti IL-4 antibody was added to aconcentration of 2 μg/ml. After 5 to 8 days IL-12 (4 ng/ml) was added tothe expansion in order to promote induction of IFN-γ producing Th1cells.

The day before transfusion to the patient the cell cultures were subjectto purification using a Ficoll-Paque PLUS (Amersham Biosciences, GEHealthcare) in order to retrieve the viable cells in the culture. On theday of transfusion the cells were washed twice in Saline solution(Natriumklorid Baxter Viaflo 9 mg/ml, Baxter) and then transferred to atransfer bag containing 100-200 ml of saline solution and 1% Human SerumAlbumin (Baxter). Investigations for microbial presence were performedprior to transfusion. Infusions of the cells were performed during 1-2hours under professional medical supervision.

Immunological Evaluation

Further immunological evaluation was performed using tritium labelledthymidine incorporation proliferation assays. An aliquot of Sentinelnode lymphocytes was set aside for this purpose, a single cellsuspension of non-sentinel node lymphocytes was obtained by gentlepressure in a loose fit glass homogenisator and peripheral bloodleukocytes were purified by Ficoll-Paque PLUS (Amersham Biosciences, GEHealthcare).

Cells were resuspended and washed twice in RPMI 1640 (Life technologies)containing 2.5% fetal calf serum (FCS) (Life technologies). Finally,cells were resuspended in RPMI 1640 proliferation media containing 10%human AB serum (Sigma), 1% penicillin-streptomycin (Sigma) and 1%glutamine (Sigma). Lymph node cells and purified PBL were used at 3×10⁵cells/well in a 96-well plate and stimulated with tumour homogenatediluted 1/100, 1/10 or Con A 10 μg/ml (Sigma) in triplicates.Proliferation was measured on day 5, 6 and 7 by adding 1 μCi of³H-Thymidine/well (Amersham) 18 hours prior to harvesting. Samples weresubjected to scintillation counting.

At the start of cell culture, stimulations of lymph node cells and PBL,for the measurement of IFN-γ secretion, were performed in 96-well plateswith 3×10⁵ cells/well in triplicate with tumour homogenate diluted 1/10and 1/100, or Con A 10 μg/ml (Sigma). The amount of secreted IFN-γ wasmeasured with ELISA (Human IFN-γ Duoset, R&D Systems) on culturesupernatants in pooled samples of the triplicates (FIG. 5). At the endof cell cultures samples of the supernatant was removed and IFN-γ andIL-4 secretion measured in triplicates with ELISA (Human IFN— Duoset andHuman IL-4 Duoset, R&D Systems) (FIGS. 6 A and 6B).

Flow Cytometry Analyses

Characterisation of cells was performed using flow cytometry initiallyon cells from the sentinel node, non-sentinel node, PBMC and from thetumour. From the sentinel node acquired lymphocytes in culture sampleswere taken every two to three weeks for flow cytometry analyses. Cellswere incubated for 30 minutes in PBS supplemented with 2% FCS and 0.05%NaN₃ (FACS buffer) with antibodies against markers for immune cellsubpopulations and for lymphocyte activation (FIGS. 3, 4 and 5).Antibodies conjugated with Fluorescein isothiocyanate (FITC) against thefollowing markers were used: CD69, HLA-DR, CD45RA, CD25, conjugated withphycoerythrin (PE): CD62L, CD19, CD45RO, CD56, conjugated withPeridinin-Chlorophyll-Protein (PerCP): CD8, CD3, conjugated withallophycocyanin (APC): CD4, CD14, CD8.

The Vβ-repertoire was examined using the Beta mark kit (BeckmanCoulter), 5×10⁵ cells/tube was stained in 10 μl of the 8 different vialscontaining mixtures of FITC, PE and dual-colour FITC-PE conjugated TCRVP antibodies and with the addition of CD8 PerCP and CD4 APC to eachtube (FIG. 7).

Example 2 Treatment of Colon Cancer by Administering Tumour-ReactiveT-Lymphocytes

Identification and Removal of Sentinel and Metinel Lymph Nodes fromColon Cancer Patients:

Sixteen patients diagnosed with colon cancer, six woman and ten men withan average age of 62 years were studied. Patients werehistopathologically classified as Duke's C or D. There were also 5patients with Duke's B with aggressive tumour characteristics such asulcerations, vascular or perineural invasion. Patients 7 and 14 howeverhad earlier been surgically treated due to colon cancer and now hadrecurrent disease with metastases to the liver. The local ethicalcommittee approved the study and each patient gave informed consent.

Identification of sentinel or metinel nodes was done intraoperatively.Mobilisation of the colonic tumour site was achieved by division ofperitoneal adhesions in order to facilitate inspection of tumour andmesentery. Injections of Patent blue dye (Guerbet, Paris) weredistributed superficially in the serosa around the tumour. Within fiveminutes, one to three mesenteric lymph nodes were coloured blue, thesesentinel nodes were marked with sutures and removed when the resectionwas complete. One non-sentinel mesenteric lymph node, distant from thetumour, was handled the same way.

The sentinel- and non-sentinel lymph nodes were cut in half and 1 mmthick slices were taken from the centre and the periphery. The rest ofthe lymph nodes were sent for histopathological examination according toroutine procedure. A piece of the tumour, including a part of theinvasive margin, was used for antigen preparation.

The lymphocytes obtained from the lymph nodes were then expanded asdescribed in Example 1.

Administration of Tumour-Reactive T-Lymphocytes:

16 patients were treated with infusion of autologous lymphocytesexpanded as described in Example 1. On average 74.7 million activatedand clonally expanded T cells were administered as a transfusion. Notoxic side effects like fever, chills, malaise, severe fluid retention,pulmonary oedema or respiratory distress were observed.

Follow-Up Evaluations

Follow-up included clinical examination every third to sixth month andcontrol of CEA levels. All stage III and IV patients were in additioninvestigated with computer tomography of the thorax and abdomen. Thepatients were followed at regular visits on average for 13 months (range5-20), median follow-up time was 13½ months. Out of the 16 patients whohad been treated with infusion of autologous lymphocytes eight had knowndistant metastases at diagnosis. Four patients received theirtransfusions due to known recurrences and out of them three are stillwithout signs of recurrences. One patient was operated due to a solitaryliver metastases and has since then been without relaps. As it appearsfrom FIG. 8 and FIG. 9, one patient with liver metastases located inboth lobes (which had been declared incurable by liver surgery) hadtotal regress of liver metastases after transfusion of tumour-reactivelymphocytes, and furthermore had normalisation of CEA levels,disappearance of ascites and is physically well fit, and exercisingregularly. One further patient with liver metastases had regress ofliver metastases and ascetic fluid after transfusion (see FIGS. 10, 11and 12). One patient had three months after transfusion regress ofmetastases in the liver and lungs (see FIGS. 13, 14, 15, and 16) withalmost a normalised CEA level at 5.9 (Normal<4.0), disappearance ofascites and he appears clinically healthy.

Results

Sixteen patients with colon cancer or solitary colorectal livermetastases were operated on at the South Stockholm General Hospital andincluded in the study. The primary locations of the tumours were threein caecum, 4 in colon ascendens, 1 in colon descendens, 7 in the sigmoidcolon and 1 in rectum. Seven right-sided hemicolectomies, 1 left-sidedhemicolectomy, 7 sigmoid resections and 1 rectumamputation wereperformed. Two patients had earlier been operated on withrectumamputation and sigmoid resection; they now underwent partial liverresections due to liver metastases. One patient had recurrences at twoabdominal locations and had earlier been operated due to a tumour in thecaecum. At our operation two sentinel nodes draining the metastasis wereidentified, one in the colonic mesentery and one in the mesentery of thesmall intestine. An extended resection of the anastomotic ileocolonicregion with mesentery was done.

In all patients, one to three (average 2.1) sentinel node(s) wereidentified intraoperatively by peritumoural patent blue injections.Among the patients with primary colonic resection on average 15.8 lymphnodes were retrieved from each specimen. After histopathologicalinvestigation of these lymph nodes five patients were classified asDuke's C and 5 patients as Duke's B, all of them were classified ashigh-risk tumours due to growth of tumour cells along nerves and invessels at pathological anatomical investigation. Five patients haddistant metastases and were at time of metastatic resection classifiedas Duke's D. Two patients of them had solitary liver metastases. Inaddition sentinel nodes were also analysed by FACS (Fluorescenceactivated cell sorter) and antibodies against cytokeratin 20, which isexpressed by colon cancer tumours, for the purpose to detectmicrometastases. The cytokeratin 20 assessments of lymph nodes by flowcytometry were in agreement with the pathological anatomical diagnosis(not shown) except in one case where a false negative sentinel node(according to histopathological analysis) was positive in thecytokeratin 20 FACS analysis.

The sentinel node is the first lymph node draining the tumour and istherefore the first site of lymph node metastasis (Dahl et al), but thesentinel node is also the primary site for the activation of the immunesystem. Tumour cells, debris, necrotic cells and antigen presentingcells accumulate in the sentinel node where presentation, activation andclonal expansion of T cells directed against the tumour occur. Thepresent inventors took advantage of this population of in vivo expandedT cell population of sentinel node acquired lymphocytes for in vitrocell culture, expansion and transfusion.

Sentinel node acquired lymphocytes is a population of T cells activatedand clonally expanded against tumour antigens that can efficiently beharvested during the surgical procedure. In contrast to recentimmunotherapy trials focusing on cytotoxic T cells, the aim of thepresent inventors was to create a protocol for in vitro enhancement ofthe in vivo initiated clonal expansion of T helper cells. T helper cellsseem to be necessary for the effective function of cytotoxic T cells andfor the creation of memory cells. Furthermore, in a T cell receptortransgenic system targeting an islet cell antigen, the transfusion ofTh1 cells was found to be sufficient for the β cell destruction anddevelopment of diabetes mellitus. In vitro culture of sentinel nodeacquired lymphocytes resulted in a Th1 activation and clonal expansionof T helper cells as indicated by the dominant production of thehallmark Th1 cytokine IFN-γ and the enrichment of a restricted TCR Vβrepertoire. The tumour homogenate used to expand the T cells is likelyto be endocytosed and processed by antigen presenting cells for class IIpresentation leading to activation of CD4⁺ T helper cells resulting inexpansion favouring T helper cells. By cross presentation antigens takenup by endocytosis may be processed and presented in the class I pocketresulting in activation of CD8⁺ cytotoxic T cells. Interestingly, insome cases the inventors found clonal expansion of both CD4⁺ and CD8⁺ Tcells.

The average number sentinel node acquired lymphocytes at start ofexpansion was 107.4 million cells (range 3.6-509 millions, median 70millions). Cells were characterised by flow cytometry. The ratio betweenCD4⁺ and CD8⁺ cells at start was in average 4.9 (range 0.36-10, median5.4) indicating an expansion CD4⁺ T helper cells in sentinel nodescompared to the CD4/CD8 ratio in peripheral blood (normal range 1.0-2.5)(FIG. 2A). In addition B lymphocytes (CD 19) and natural killer (NK)cells (CD 56) were present in sentinel nodes (not shown). The cells wereheld in culture in average 36.1 days (range 23-58 days), median 33 days.Cells were monitored closely by flow cytometry at least weekly.Initially the total number of cells decreased. B cells and NK cellsdisappeared almost completely and the number of CD8⁺ T killer cells wasdiminished. The culture procedure used promoted mainly the expansion ofT helper cells since the average CD4/CD8 ratio was 92.5. Restimulationwith autologous tumour antigen resulted in clonal expansion of tumourreactive T cells as assessed by investigating the T cell receptor Vβrepertoire of sentinel node acquired lymphocytes before and after invitro culture.

Before transfusion expanded T cells were functionally tested againstautologous tumour antigens by measuring activation and cytokineproduction of the Th1 cytokine IFN-γ and the Th2 cytokine IL-4. In vitroexpanded sentinel node acquired lymphocytes responded upon restimulationwith tumour antigen with the production of IFN-g and no or very littleIL-4 indicating that the expanded T cells were functional and Th1responsive.

Six patients with Duke's D were treated in the study. Two patientsstaged as Duke's D at surgery with metastases to the liver and to thelungs and liver, respectively displayed marked regression of disease(pat 5 and 12). After transfusion of lymphocytes the first patient hadtotal regress of liver metastases located in both lobes (which had beendeclared incurable by liver surgery) (FIG. 3) normalisation of CEAlevels, disappearance of ascites and appear healthy. Patient 12 showsregress of metastases in the liver and lungs with almost a normalisedCEA level at 5.9 (Normal<4.0), disappearance of ascites and he appearsclinically healthy. Patient 1 displayed a regression of the size ofliver metastasis, and initially a decrease in CEA levels, disappearanceof ascites and she was in excellent shape when she suddenly died (day191), what appears to have been a lung embolus. Two Duke's D patientsdisplay stable disease without progression of metastasis or increase inCEA levels. The oldest patient no 7 in the study displayed stabledisease for five months, but thereafter CEA levels started to increaseand she died at age 83. No autopsy was performed. One patient was stagedas Duke's C at surgery but soon developed metastases to the liver andlungs (Duke's D), but following transfusion and chemotherapy a regressof the lung and liver metastases were seen with only slightly elevatedCEA levels. The patients classified as Duke's C all have normal CEAlevels and appear without any signs of radiological or clinicalrecurrence of disease. Four of the Duke's B patients are healthy withnormal CEA levels and have no signs of recurrent disease. Patient no 9classified as Duke's B, but with an aggressive growing tumour showssigns of recurrent disease with elevated CEA levels (67) and signs ofliver metastases.

To investigate the fate of transfused T cells the present inventorsanalysed T cell proliferation against tumour extract in peripheralblood. As mentioned before, they could not demonstrate any T cellreactivity in peripheral blood against autologous tumour antigens in anyof the patients prior to transfusion. However, we were able to detect Tcell proliferation against autologous tumour antigens in peripheralblood in all investigated patients up to 10 months after transfusionindicating the presence of clonally expanded circulating tumour-reactiveT cells.

Summary of Patient Characteristics

Below is a table of all participants in the study, sorted after Duke'sclassification at surgery:

Participant characteristics Duke's Overall Age/ Classi- Infused CD4/IFN-y survival Sex fication cells (×10⁶) CD8 ^(a) (pg/ml) (months)Response 67/M B 4  92/0.2 ND 31 SD 67/F B 8 15/51 ND 30 SD 71/M B 5074/15 2091 29 SD 74/M B 63 64/22 ND 29 SD 66/M B 152  82/1.5 1411 27 SD64/F C 110 64/25 ND 34 SD 58/F C 16 77/18 417 23 SD 61/F D 1 3.7/35  ND6 SD 47/M D 80 24/16 ND 36 CR 54/M D 40 37/24 ND 36 SD 65/M D 270 82/15ND 36 CR 42/F D 80 66/11 ND 33 CR 82/F D 40  98/0.1 ND 6 SD 74/M D 13073/22 142 30 CR 33/M D 72  72/1.5 908 12 PR 66/M D 25 37/27 764 26 PR^(a)The numbers represent the percentage of CD4 and CD8 positive cellsdetected with FACS.Discussion

To the knowledge of the present inventors, sentinel or metinelnode-based immunotherapy in patients with colon cancer has never beenpresented before. Thus, this is the first attempt to use lymphocytesacquired from sentinel or metinel nodes for therapy. There are somemajor differences between the present study and e.g. treatment withhigh-dose IL-2 (Rosenberg). Firstly the use of sentinel node acquiredlymphocytes that have been in vitro stimulated by autologous tumourhomogenate and APCs, causes a highly specific cellular immune responsetowards the tumour. Only T cells with high affinity to the primarytumour will survive until transfusion. In a systemic generalizedtreatment with high-dose IL-2 intravenously to patients all lymphocyteswill be equally stimulated and reasonably only a very small fraction ofthem are tumour specific. The present inventors believe that since thesentinel node(s) are the first draining lymph nodes to a tumour therewill be an excessive accumulation of tumour specific lymphocytes. Theproliferation and transfusion of true tumour recognising T cells shouldcreate a massive tumour specific reaction. Secondly the high-dose IL-2regimen causes high-toxicity and severe complications, long treatmentperiods and high costs. The transfusions according to the present methodhave been given without complications during about one hour and thepatients are often discharged the same day. Thirdly, the presentprotocol aim towards expansion of T helper cells from sentinel nodes, incontrast to expansion of cytotoxic T cells harvested as tumourinfiltrating lymphocytes.

This study shows that freshly isolated sentinel node acquiredlymphocytes possesses a proliferative ability in vitro againstautologous tumour homogenate and can without complications be transfusedto the patient as adoptive immunotherapy. There is a strong indicationto that treatment with expanded sentinel node acquired lymphocytes mayimprove the outcome of patients with high-risk or disseminated coloncancer, as well as patients suffering from types of solid cancer.

Example 3 Expansion of Lymph Node Acquired Lymphocytes from Patient withGallbladder Cancer

On day one, cell cultures containing approximately 35 million(Culture 1) and 5 million cells (Culture 2) was set up.

Throughout the whole culturing period, cells were kept in incubator at37° C. and 5% CO₂ and 240 IU IL-2/mL media added every 3-4 days.

Initially, both cultures contained a high proportion of red blood cellswhich eventually decreased as only lymphocytes proliferated in thecultures.

On day 3, autologous tumor extract were added to both cultures at aconcentration of 1/100.

On day 10, phase II was initiated in both cultures by adding radiatedantigen presenting cells, autologous tumor extract and interleukin-2(240 IU/ml) to the cultures. At this stage, the cultures containedapproximately 45 million and 15 million lymphocytes, respectively.

Cultures were maintained regularly by keeping the cell concentration atapproximately 3 million cells/ml.

Cells from both cultures were harvested on day 25 and a cell transfusionsolution containing cells from both cultures was prepared.

The final number of cells were 94 million for Culture 1 and 79 millionfor Culture 2. Culture 1 contained approximately 85% CD4 T cells and 5%CD8 T cells.

V beta profiling of Culture 1 revealed high proportions of V betaprofiles 1, 2 and 22, indicating that clonal expansions of cells withthese profiles had occurred.

Example 4 Expansion of Sentinel Node Acquired Lymphocytes from Patientwith Small Intestine Cancer

On day one, lymphocytes were extracted from three sentinel lymph nodesand put into separate cultures (Culture 1-3).

Main conditions of the cell cultures were as described in example 3.

The cultures initially contained 27, 37, and 6 million cells,respectively.

Analysis of cultures on day 2 revealed CD4/CD8 ratios in the cultures of79% CD4/8% CD8 for Culture 1, for Culture 2 46%/8%, and for Culture 327%/4%.

Early analysis revealed the Cultures 2 and 3 both had strong tumourpresence. However, at the end of culture both cultures were free oftumour cells, indicating that only T cells had proliferated in thesecultures.

On day 3, autologous tumor extract were added to all cultures at aconcentration of 1/100.

On day 15, phase II were initiated in all cultures by adding radiatedantigen presenting cells, autologous tumor extract and interleukin-2(240 IU/ml) to the cultures. At this stage, Culture 1 and 2 containedapproximately 50 million cells and Culture 3 about 30 million cells.

At beginning of phase II, the CD4/CD8 ratios were 74%/10% in Culture 1,66%/11% in Culture 2 and 77%/7% in Culture 3.

On day 25, the cells from all cultures were harvested and a celltransfusion solution containing cells from all cultures was prepared.The total final number of cells in all cultures was 136 million.

Analysis of CD4/CD8 ratios was done the day before harvest, with thefollowing results:

Culture 1: 59%/21%, Culture 2: 58%/24%, and Culture 3: 57%/14%. Theresults of the analysis indicated that both CD4 and CD8 T cells hadproliferated in Phase II.

Example 5 Expansion of Sentinel Node Acquired Lymphocytes from Patientwith Mesothelioma

Lymphocytes were acquired from four different Sentinel Nodes draining amesothelioma tumor originating in pleura of a patient. On day one, oneculture from each sentinel node was set up.

Throughout the whole culturing period, cells were kept in incubator at37° C. and 5% CO₂ and 240 IU IL-2/mL media added every 3-4 days.

Initial data for cell number and CD4/CD8 ratios were as follows:

Culture 1: 39 million 18% CD4/4% CD8 Culture 2: 97 million 25%/5%Culture 3: 32 million 26%/8% Culture 4: 17 million 28%/9%

Initial analysis of showed presence of tumor cells in all cultures(10-15%). Especially Cultures 3 and 4 showed strong presence of tumorcells. However, during the course of culturing all cultures presenteddecreased tumor cell presence. At day 21 tumor presence had decreased to0.5-1.5% in all four cultures, indicating that only T cells hadproliferated in the cultures.

On day 3, autologous tumor extract were added to all cultures at aconcentration of 1/100.

On day 14, phase II were initiated in all cultures by adding radiatedantigen presenting cells, autologous tumor extract and interleukin-2(240 IU/ml) to the cultures.

Cell count and analysis of T cell characteristics was done on day 21 forall cultures, revealing the following data:

Culture 1: 200 million 63% CD4/21% CD8 Culture 2: 350 million 77%CD4/13% CD8 Culture 3: 350 million 57% CD4/28% CD8 Culture 4: 350million 49% CD4/34% CD8

Results indicate expansion of both CD4 and CD8 cells in all cultures.

Analysis of the Vbeta profiles of Cultures 1 and 2 was performed on day2 and on day 24 of the cell culturing period (see FIGS. 12 and 13).Results of the analysis clearly indicate that a clonal expansion of Tcells belonging to Vbeta groups 4 and 14 had taken place. In culture 1,Vbeta group 4 had been expanded by a factor 4 and Vbeta group 14 hadbeen expanded by a factor 6. Interestingly, a similar general pattern ofclonal expansion pattern was seen in both cultures.

Specific Embodiments

The following is a list of specific embodiments of the presentinvention. It is to be understood that such embodiments are presented asexemplary only and that the present invention is to in no way be limitedthereby.

1. A method for treating a patient suffering from a neoplastic diseaseselected from gall bladder cancer, hepato cellular cancer, ovariancancer, small intestine cancer, lung cancer, mesothelioma, breastcancer, kidney cancer, pancreas cancer, prostate cancer, carcinoidcancer, leiomyosarcoma, or metastasis thereof, the method comprising

-   -   i) identifying in a patient one or more sentinel and/or metinel        lymph nodes draining a neoplasm selected from gall bladder        cancer, hepato cellular cancer, ovarian cancer, small intestine        cancer, lung cancer, mesothelioma, breast cancer, kidney cancer,        pancreas cancer, prostate cancer, carcinoid cancer,        leiomyosarcoma, or metastasis thereof,    -   ii) resecting the one or more nodes and, optionally all or part        of the tumour or metastasis,    -   iii) isolating tumour-reactive T-lymphocytes from said lymph        nodes,    -   iv) in vitro expanding said tumour-reactive T-lymphocytes,    -   v) administering the thus obtained tumour-reactive T-lymphocytes        to the patient,        wherein the T-lymphocytes are CD4+ helper and/or CD8+        T-lymphocytes.        2. A method according to embodiment 1 or 2, wherein the one or        more lymph nodes are identified in step i) by injecting one or        more lymph node locators into the patient.        3. A method according to any of the preceding embodiments,        wherein the one or more lymph node locators are affinity based.        4. A method according to any of the preceding embodiments,        wherein the one or more lymph node locators are non-affinity        based.        5. A method according to any of the preceding embodiments        wherein one or more lymph node locators are injected into,        above, around, adjacent and/or under the tumour or metastasis.        6. A method according to any of the preceding embodiments,        wherein the one or more lymph node locators are injected by a        single injection.        7. A method according to any of embodiments 1-5, wherein the one        or more lymph node locators are injected by multiple injections.        8. A method according to any of the preceding embodiments,        wherein the one or more lymph node locators are injected by a        non-surgical procedure.        9. A method according to any of embodiments 1-79, wherein the        one or more lymph node locators are injected as part of a        surgical procedure.        10. A method according to any of the preceding embodiments,        wherein all or part of the tumour or metastasis is removed from        the patient.        11. A method according to any of the preceding embodiments        wherein the in vitro expansion step iv) comprises    -   i) a first phase of stimulating tumour-reactive CD4+ helper        and/or CD8+ T-lymphocytes with tumour-derived antigen together        with at least one substance having agonistic activity towards        the IL-2 receptor to promote survival of tumour-reactive CD4+        helper and/or CD8+ T-lymphocytes, and    -   ii) a second phase of activating and promoting growth of        tumour-reactive CD4+ helper and/or CD8+ T-lymphocytes, wherein        the second phase ii) is initiated when the CD25 cell surface        marker (or IL-2R marker) is down-regulated on T-lymphocytes.        12. A method according to embodiment 11, wherein the        down-regulation is defined as that 5% or less of the        T-lymphocyte population expresses CD25.        13. A method according to embodiment 11 or 12, wherein the        T-lymphocytes are present in a culture medium.        14. A method according to embodiment 13, wherein the culture        medium is a serum-free medium, such as, e.g. AIMV medium.        15. A method according to any of the preceding embodiments,        wherein the first phase i) is initiated by adding the at least        one substance having agonistic activity towards the IL-2        receptor.        16. A method according to embodiment 15, wherein the substance        having agonistic activity towards the IL-2 receptor is IL-2.        17. A method according to embodiment 16, wherein IL-2 is added        in a low dose, such as, e.g., from about 100 IU/ml culture        medium to about 700 IU/ml culture medium, from about 100 IU/ml        culture medium to about 600 IU/ml culture medium, from about 100        IU/ml culture medium to about 500 IU/ml culture medium, from        about 100 IU/ml culture medium to about 400 IU/ml culture        medium, from about 100 IU/ml culture medium to about 300 IU/ml        culture medium and from about 100 IU/ml culture medium to about        200 IU/ml culture medium.        18. A method according to any of the preceding embodiments,        wherein a further amount of the at least one substance having        agonistic activity towards the IL-2 receptor is added regularly        throughout phase i), such as, e.g., every 2^(nd), 3^(rd) or        4^(th) day of phase i).        19. A method according to embodiment 18, wherein the substance        having agonistic activity towards the IL-2 receptor is IL-2.        20. A method according to embodiment 19, wherein the        concentration of IL-2 added is from about 100 IU/ml culture        medium to about 700 IU/ml culture medium, from about 100 IU/ml        culture medium to about 600 IU/ml culture medium, from about 100        IU/ml culture medium to about 500 IU/ml culture medium, from        about 100 IU/ml culture medium to about 400 IU/ml culture        medium, from about 100 IU/ml culture medium to about 300 IU/ml        culture medium and from about 100 IU/ml culture medium to about        200 IU/ml culture medium.        21. A method according to any of the preceding embodiments,        wherein the tumour-derived antigen is added from day 2 to and        including day 5 of the first phase i), such as, e.g., on day 2,        on day 3, on day 4 or on day 5.        22. A method according to any of embodiments 11-20, wherein the        tumour-derived antigen is added essentially at the same time as        when phase i) is initiated or at the most up to 3 days        thereafter.        23. A method according to any of the preceding embodiments,        wherein the tumour-derived antigen is a denatured homogenate of        a tumour.        24. A method according to any of the preceding embodiments,        wherein the tumour-derived antigen is a protein, polypeptide or        peptide.        25. A method according to any of the preceding embodiments,        wherein the second phase ii) is initiated from day 17 to and        including day 23 of the first phase i), such as, e.g. on day 17,        on day 18, on day 19, on day 20, on day 21, on day 22 or on day        23.        26. A method according to any of the preceding embodiments,        wherein the second phase is initiated by the addition of        tumour-derived antigen to the T-lymphocytes for activating        tumour-reactive CD25-negative T-lymphocytes.        27. A method according to embodiment 26, wherein the        tumour-derived antigen is a denatured homogenate of a tumour.        28. A method according to embodiment 26, wherein the        tumour-derived antigen is a tumour protein, polypeptide or        peptide.        29. A method according to any of embodiments 26-28, which        further comprises addition to the T-lymphocytes of antigen        presenting cells together with the tumour-derived antigen.        30. A method according to embodiment 20, wherein the antigen        presenting cells are irradiated peripheral blood leucocytes        containing antigen-presenting B-cells and/or monocytes.        31. A method according to any of the preceding embodiments,        wherein the second phase ii) comprises adding at least one        substance capable of up-regulating IL-12R on the T-lymphocytes.        32. A method according to embodiment 31, wherein the        substance(s) capable of up-regulating IL-12R on the        T-lymphocytes is substance(s) having agonistic activity towards        an interferon receptor.        33. A method according to embodiment 32, wherein the        substance(s) having agonistic activity towards an interferon        receptor is an interferon.        34. A method according to embodiment 33, wherein the        substance(s) having agonistic activity towards an interferon        receptor is interferon-α.        35. A method according to any of embodiments 31-34, wherein the        substance(s) capable of up-regulating IL-12R on the        T-lymphocytes, such as, e.g. a substance having agonistic        activity towards an interferon receptor, is added when the level        of IL-12 is at least 1 fold, such as, e.g., at least 2, at least        3 fold, at least 4 fold, or at least 5 fold increased as        compared to the level of IL-12 on day 1 of phase ii).        36. A method according to any of embodiments 31-35, wherein the        substance capable of up-regulating IL-12R on the T-lymphocytes,        such as, e.g. a substance having agonistic activity towards an        interferon receptor is added from day 2 to and including day 4        after initiating the second phase ii), such as, e.g. on day 2,        on day 3 or on day 4.        37. A method according to any of the preceding embodiments,        wherein the second phase ii) comprises adding one or more        substances capable of antagonizing development of Th2 type        T-lymphocytes.        38. A method according to embodiment 37, wherein the one or more        substances capable of antagonizing development of Th2 type        T-lymphocytes are one or more substances capable of neutralizing        IL-4, IL-5, IL-10, and/or TGF-beta.        39. A method according to embodiment 38, wherein the one or more        substances capable of neutralizing IL-4, IL-5, IL-10, and/or        TGF-beta are anti IL-4 antibody, anti IL-5 antibody and/or anti        IL-10 antibody.        40. A method according to any of embodiments 37-39, wherein the        one or more substances capable of antagonizing development of        Th2 type T-lymphocytes, such as, e.g., one or more substances        capable of neutralizing IL-4, IL-5, IL-10, and/or TGF-beta is        added on day 1 of the second phase ii).        41. A method according to any of embodiments 37-39, wherein the        one or more substances capable of antagonizing development of        Th2 type T-lymphocytes, such as, e.g., one or more substance        capable of neutralizing IL-4, IL-5, IL-10, and/or TGF-beta is        added in a subsequent step after addition of the substance        capable of up-regulating IL-12R on the T-lymphocytes.        42. A method according to embodiment 41, wherein the one or more        substances capable of antagonizing development of Th2 type        T-lymphocytes, such as, e.g., one or more substance capable of        neutralizing IL-4, IL-5, IL-10, and/or TGF-beta is added one day        after addition of the substance capable of up-regulating IL-12R        on the T-lymphocytes.        43. A method according to any of the preceding embodiments,        wherein a further amount of the one or more substance capable of        antagonizing development of Th2 type T-lymphocytes, such as,        e.g., one or more substance capable of neutralizing IL-4, IL-5,        IL-10 and/or TGF-beta is added regularly throughout phase ii).        44. A method according to embodiment 43, wherein a further        amount of the one or more substance capable of antagonizing        development of Th2 type T-lymphocytes, such as, e.g., one or        more substance capable of neutralizing IL-4, IL-5, IL-10 and/or        TGF-beta is added every 2^(nd), 3^(rd) or 4^(th) day of phase        ii).        45. A method according to any of the preceding embodiments,        wherein a further amount of a substance having agonistic        activity towards the IL-2 receptor is added regularly throughout        phase ii).        46. A method according to embodiment 45, wherein the substance        having agonistic activity towards the IL-2 receptor is added        every 2^(nd), 3^(rd) or 4^(th) day of phase ii), such as, e.g.,        every 3^(rd) day.        47. A method according to embodiment 45 or 46, wherein the        substance having agonistic activity towards the IL-2 receptor is        IL-2.        48. A method according to any of the preceding embodiments,        wherein the second phase ii) comprises adding one or more        substances promoting the development of Th1 type T-lymphocytes.        49. A method according to embodiment 48, wherein the one or more        substances promoting the development of Th1 type T-lymphocytes        is substances having agonistic activity towards the IL-7, IL-12,        IL-15 and/or IL-21 receptor.        50. A method according to embodiment 49, wherein the one or more        substances is selected from IL-7, IL-12, IL-15 and IL-21.        51. A method according to any of embodiments 48-50, wherein one        or more substances promoting the development of Th1 type        T-lymphocytes, such as, e.g., substances having agonistic        activity towards the IL-7, IL-12, IL-15 and/or IL-21 receptor is        added when the level of IFN-gamma is increased as compared to        the level of IFN-gamma on initiation of second phase ii).        52. A method according to embodiment 51, wherein the increased        level of IFN-gamma is determined as at least a 1 fold increase        in IFN-gamma level, such as, e.g., at least a 2 fold, at least a        3 fold, at least a 4 fold increase as compared to the level of        IFN-gamma on initiation of the second phase ii).        53. A method according to any of embodiments 48-52, wherein the        one or more substances promoting the development of Th1 type        T-lymphocytes, such as, e.g., substances having agonistic        activity towards the IL-12, IL-15 and/or IL-21 receptor is added        when CD25 and/or CD69 are down-regulated.        54. A method according to any of embodiments 48-53, wherein the        concentration of each of the one or more substances promoting        the development of Th1 type T-lymphocytes, such as, e.g.,        substances having agonistic activity towards the IL-7, IL-12,        IL-15 and/or IL-21 receptor added is from about 150 IU/ml        culture medium to about 300 IU/ml culture medium, such as, e.g.        250 IU/ml culture medium.        55. A method according to any of embodiment 48-54, wherein the        one or more substances promoting the development of Th1 type        T-lymphocytes, such as, e.g., substances having agonistic        activity towards the IL-12, IL-15 and/or IL-21 receptor is added        from day 5 to and including day 8 after initiating the second        phase ii), such as, on day 5, day 6, day 7 or day 8.        56. A method according to any of the preceding embodiment for        the preparation of CD4+ helper T lymphocytes.        57. A method according to any of the preceding embodiments for        the preparation of effector T-lymphocytes.        58. A method according to any of the preceding embodiments for        the preparation of memory T-lymphocytes.        59. A method according to any of the preceding embodiments for        the preparation of Th1 type T-lymphocytes.        60. A method according to any of the preceding embodiments,        which further comprises monitoring the expression of cell        surface markers, such as, e.g., CD25 and/or CD69 on the        T-lymphocytes continuously during the first phase i) and second        phase ii).        61. A method according to embodiment 60, wherein the        T-lymphocytes are harvested when CD25 on T-lymphocytes in the        second phase ii) is down-regulated.        62. A method according to embodiment 61, wherein the        T-lymphocytes are subjected to at least one additional round of        phase ii), when CD25 on T-lymphocytes is down-regulated.        63. A method according to embodiment 61 or 62, wherein the        down-regulation is defined as that 5% or less of the CD4        positive T-lymphocyte population expresses CD25.        64. A method according to any of the preceding embodiments,        wherein the tumour-reactive T-lymphocytes are harvested from day        10 to and including day 14 after initiating the second phase        ii).        65. A method according to embodiment 64, wherein the        tumour-reactive T-lymphocytes are purified after harvest.        66. A method according to any of the preceding embodiments        further comprising a step of freezing the tumour-reactive        T-lymphocytes obtained in the second phase ii).        67. A method according to any of the preceding embodiments,        wherein the T-lymphocytes are derived from lymph nodes draining        a primary tumour and/or a metastasis, or they are derived from        blood.        68. A method according to any of the preceding embodiments        further comprising a step of freezing the tumour-reactive        T-lymphocytes obtained in the second phase ii).        69. A method according to any of the preceding embodiments,        wherein the T-lymphocytes are derived from lymph nodes draining        a primary tumour and/or a metastasis, or they are derived from        blood.        70. A method according to any of the preceding embodiments,        wherein the tumour-reactive T-lymphocytes in step iv) are        administered intravenously, intraarterially or intrathecally,        intraperitonally.        71. A method according to any of the preceding embodiments,        wherein the amount of tumour-reactive T-lymphocytes administered        is at least 10 million, such as, e.g. at least 20 million, at        least 30 million, at least 40 million, at least 50 million, at        least 60 million, at least 70 million or at least 80 million.        72. A method according to any the preceding embodiments, wherein        the tumour-reactive T-lymphocytes administered are a combination        of effector T-lymphocytes and memory T-lymphocytes.        73. A method according to embodiment 72, wherein the percentage        of effector T-lymphocytes is from about 10% to about 65%, such        as, e.g., from about 20% to about 50% or from about 30% to about        40%.        74. A method according to any of the preceding embodiments,        wherein the tumour-reactive T-lymphocytes are autologous.        75. A method according to any of the preceding embodiments,        wherein the tumour-reactive T-lymphocytes are non-autologous.        76. A tumour-reactive T-lymphocyte prepared according to the        method defined in any of embodiments 1-75.        77. A tumour-reactive T-lymphocyte according to embodiment 76,        which is a CD4+ T-lymphocyte.        78. A tumour-reactive T-lymphocyte according to embodiment 76 or        77, which is an effector T-lymphocyte.        79. A tumour-reactive T-lymphocyte according to any of        embodiments 76-78, which is a memory T-lymphocyte.        80. A tumour-reactive T-lymphocyte according to any of        embodiment 76-79, which is a Th1 type T-lymphocyte.        81. Use of tumour-reactive T-lymphocytes according to any of        embodiments 76-80, for the preparation of a medicament for the        treatment of disseminated cancer.        82. Kit for use in a method according to any of embodiments        1-75, the kit comprising a media for cultivation of        T-lymphocytes.        83. Kit according to embodiment 82 further comprising one or        more substances for stimulating, activating and directing        tumour-reactive T-lymphocytes.        84. Kit according to embodiment 82 or 83, wherein the media a        serum free medium, such as, e.g. AIMV, RPMI 1640, DMEM or MEM.        85. Kit according to any of embodiments 82-84, wherein the one        or more substances for stimulating, activating an directing        tumour-reactive T-lymphocytes are selected from tumour-derived        antigen, substances having agonistic activity towards the IL-2        receptor, substances capable of up-regulating IL-12R on the        T-lymphocytes, substances capable of antagonizing development of        Th2 type T-lymphocytes and substances promoting the development        of Th1 type T-lymphocytes.        86. Kit according to any of embodiments 82-85, wherein the one        or more substances for stimulating, activating and directing        tumour-reactive T-lymphocytes are selected from the group        comprising IL-2, interferon-alpha, anti-IL-4 antibody, anti-IL-5        antibody, anti-IL-10 antibody, IL-7, IL-12, IL-15 and IL-21.        87. Kit according to any of embodiments 82-86, comprising a        pharmaceutical composition suitable for intravenous        administration.        88. A kit for detection of sentinel or metinel lymph nodes, the        kit comprising a syringe and a lymph node locator.        89. A kit for detection of sentinel or metinel lymph nodes, the        kit comprising a syringe prefilled with a lymph node locator.        90. Kit according to any of embodiments 82-89 further comprising        instructions for use.        91. Kit according to embodiment 90, wherein the instructions are        in the form of computer software.

1. A method for treating a patient suffering from a neoplastic diseaseselected from gall bladder cancer, hepato cellular cancer, ovariancancer, small intestine cancer, lung cancer, mesothelioma, breastcancer, kidney cancer, pancreas cancer, prostate cancer, carcinoidcancer, leiomyosarcoma, or metastasis thereof, the method comprising: a)isolating tumour-reactive T-lymphocytes from either the patient's blood,or from the patient's lymph nodes, wherein tumour-reactive T-lymphocytesisolated from the patient's lymph nodes are obtained by: i) identifyingin a patient one or more sentinel and/or metinel lymph nodes draining aneoplasm selected from gall bladder cancer, hepato cellular cancer,ovarian cancer, small intestine cancer, lung cancer, mesothelioma,breast cancer, kidney cancer, pancreas cancer, prostate cancer,carcinoid cancer, leiomyosarcoma, or metastasis thereof, ii) resectingthe one or more nodes and, optionally all or part of the tumour ormetastasis, and iii) isolating tumour-reactive T-lymphocytes from saidlymph nodes; b) in vitro expanding said tumour-reactive T-lymphocytesisolated from either the patient's blood or lymph nodes using: i) afirst phase of stimulating tumour-reactive CD4+ helper and/or CD8+T-lymphocytes with tumour-derived antigen together with at least onesubstance having agonistic activity towards an IL-2 receptor to promotesurvival of tumour-reactive CD4+ helper and/or CD8+ T-lymphocytes; andii) a second phase of activating and promoting growth of tumour-reactiveCD4+ helper and/or CD8+ T-lymphocytes, wherein the second phase isinitiated when a CD25 cell surface marker or IL-2R marker isdown-regulated on T-lymphocytes; and c) administering the thus obtainedtumour-reactive T-lymphocytes to the patient.
 2. A method according toclaim 1, wherein the one or more lymph nodes are identified by injectingone or more lymph node locators into the patient.
 3. A method accordingto claim 2, wherein the one or more lymph node locators are affinitybased.
 4. A method according to claim 2, wherein the one or more lymphnode locators are non-affinity based.
 5. A method according to claim 2,wherein one or more lymph node locators are injected into, above,around, adjacent and/or under the tumour or metastasis.
 6. A methodaccording to claim 1, wherein the down-regulation is defined as that 5%or less of the T-lymphocyte population which expresses CD25.
 7. A methodaccording to claim 1, wherein the first phase is initiated by adding theat least one substance having agonistic activity towards the IL-2receptor.
 8. A method according to claim 1, wherein the tumour-derivedantigen is a denatured homogenate of a tumour.
 9. A method according toclaim 1, wherein the second phase is initiated by the addition oftumour-derived antigen to the T-lymphocytes for activatingtumour-reactive CD25-negative T-lymphocytes.
 10. A method according toclaim 1 or 9, which further comprises addition to the T-lymphocytes ofantigen presenting cells together with the tumour-derived antigen.
 11. Amethod according to claim 10, wherein the antigen presenting cells areirradiated peripheral blood leucocytes containing antigen-presentingB-cells and/or monocytes.
 12. A method according to claim 1, wherein thesecond phase comprises adding at least one substance capable ofup-regulating IL-12R on the T-lymphocytes.
 13. A method according toclaim 1, wherein the second phase comprises adding one or moresubstances capable of antagonizing development of Th2 typeT-lymphocytes.
 14. A method according to claim 13, wherein the one ormore substances capable of antagonizing development of Th2 typeT-lymphocytes are one or more substances capable of neutralizing IL-4,IL-5, IL-10, and/or TGF-beta.
 15. A method according to claim 14,wherein the one or more substances capable of neutralizing IL-4, IL-5,IL-10, and/or TGF-beta are anti IL-4 antibody, anti IL-5 antibody and/oranti IL-10 antibody.
 16. A method according to claim 13, wherein the oneor more substances capable of antagonizing development of Th2 typeT-lymphocytes, is added on day 1 of the second phase.
 17. A methodaccording to claim 1, wherein the second phase comprises adding one ormore substances promoting the development of Th1 type T-lymphocytes. 18.A method according to claim 17, wherein the one or more substancespromoting the development of Th1 type T-lymphocytes is substances havingagonistic activity towards the IL-7, IL-12, IL-15 and/or IL-21 receptor.19. A method according to claim 18, wherein the one or more substancesis selected from IL-7, IL-12, IL-15 and IL-21.
 20. A method according toclaim 1, for the preparation of CD4+ helper T lymphocytes.
 21. A methodaccording to claim 1, for the preparation of effector T-lymphocytes. 22.A method according to claim 1, for the preparation of memoryT-lymphocytes.
 23. A method according to claim 1, for the preparation ofTh1 type T-lymphocytes.
 24. A method according to claim 1, which furthercomprises monitoring the expression of cell surface markers, on theT-lymphocytes continuously during the first phase and second phase. 25.A method according to claim 24, wherein the T-lymphocytes are harvestedwhen CD25 on T-lymphocytes in the second phase is down-regulated.
 26. Amethod according to claim 1, further comprising a step of freezing thetumour-reactive T-lymphocytes obtained in the second phase.
 27. A methodaccording to claim 1, wherein the T-lymphocytes are derived from bloodand wherein the tumour-derived antigen is added essentially at the sametime as when phase is initiated or at the most up to 2 days thereafter.28. A method according to claim 1, wherein the tumour-reactiveT-lymphocytes are administered to the patient intravenously,intraarterially or intrathecally, intraperitonally.
 29. A methodaccording to claim 1, wherein the amount of tumour-reactiveT-lymphocytes administered is at least 10 million.
 30. A methodaccording to claim 1, wherein the tumour-reactive T-lymphocytesadministered are a combination of effector T-lymphocytes and memoryT-lymphocytes.
 31. A method according to claim 30, wherein thepercentage of effector T-lymphocytes is from about 10% to about 65.